Preinitiation Complex Formation Research Articles

General Regulatory Factors Control the Fidelity of Transcription by Restricting Non-coding and Ectopic Initiation

The fidelity of transcription initiation is essential for accurate gene expression, but the determinants of start site selection are not fully understood. Rap1 and other general regulatory factors (GRFs) control the expression of many genes in yeast. We show that depletion of these factors induces widespread ectopic transcription initiation within promoters. This generates many novel non-coding RNAs and transcript isoforms with diverse stability, drastically altering the coding potential of the transcriptome. Ectopic transcription initiation strongly correlates with altered nucleosome positioning. We provide evidence that Rap1 can suppress ectopic initiation by a "place-holder" mechanism whereby it physically occludes inappropriate sites for pre-initiation complex formation. These results reveal an essential role for GRFs in the fidelity of transcription initiation and in the suppression of pervasive transcription, profoundly redefining current models for their function. They have important implications for the mechanism of transcription initiation and the control of gene expression.

MGP Interacts with BMP-4 and BMP-2 and Supports Normal and Malignant Hematopoietic Stem/Progenitor Cells

The general transcriptional coregulatory complex Mediator, subcomplex of the RNA polymerase II holoenzyme, is the endpoint convergence of a variety of intracellular signals, and initiates transcription through recruitment of general initiation factors and formation of a functional preinitiation complex. Among the Mediator subunits, MED1 is crucial for hematopoiesis: it is known to be employed in transcription of genes involved in hematopoietic niche function, as well as in differentiation of hematopoietic precursor cells (e.g., RARα- and VDR-mediated myelomonopoiesis, GATA1-mediated erythromegakaryopoiesis, and iNKT cell development). Among the attenuated genes in Med1-/- mesenchymal stromal cells was matrix Gla protein (MGP), modulator of the BMP-SMAD signals. As MGP is abundantly expressed in bone tissues and reportedly interacts with niche factors BMP-4 and BMP-2, we hypothesized that MGP might modulate niche function in hematopoiesis through the interaction with these BMPs. We tested this possibility by an in vitro niche model composed of a coculture of bone marrow (BM) mesenchymal stromal cells and BM hematopoietic cells. When mouse BM hematopoietic cells were cocultured with OP-9 or MS-5 mouse BM stromal cells in the presence of the blocking antibody against MGP, the growth and DNA synthesis of BM hematopoietic cells were attenuated and the number of long-term culture-initiating cells (LTC-ICs), which corresponded to the supported hematopoietic stem/progenitor cells (HSPCs), decreased. We next asked if MGP also functioned as a niche factor for malignant HSPCs, To this end, we utilized MB-1 cells, blast crisis CML-derived myeloblastic leukemia cells. MB-1 cells are dependent on cocultured BM stromal cells and possess characteristics of leukemia stem cells (LSCs). When MB-1 cells were cocultured with OP-9 or MS-5 BM stromal cells in the presence of the anti-MGP antibody, the number of MB-1 cells, as well as the number of cobblestone formation, i.e., LSC nature, of MB-1 cells, was profoundly attenuated. Therefore, MGP appears to have a trophic effect on hematopoietic cells and to support normal and malignant HSPCs in our niche model. MGP was, both at mRNA and protein levels, expressed abundantly in BM stromal cells but scarcely in normal hematopoietic cells. However, MB-1 leukemia cells (presumably ectopically) expressed a meaningfully high level of MGP. During the coculture of normal BM hematopoietic cells with OP-9 or MS-5 BM stromal cells, MGP was induced prominently and transiently after one day. MGP, expressed in MS-5 or OP-9 BM stromal cells, was likewise transiently induced after 1 day of culture with BM hematopoietic cells that were physically dissociated from these BM stromal cells by the transwell apparatus, indicating that secreted humoral factor(s) induced the MGP expression in the BM stromal cells. As for a malignant situation, the expressions of MGP in both MB-1 leukemia cells and OP-9 or MS-5 BM stromal cells were fluctuated in an oscillated days period. Since the support of HSPCs required their association with BM stromal cells, the induced MGP expression was apparently insufficient for HSPCs support, and the action of induced MGP may be indirect. Therefore, we next asked if BMP-4 and BMP-2, which reportedly associate with MGP, were also induced by the coculture and were employed in HSPCs support jointly with MGP. Indeed, during the coculture, BMP-4 was rapidly and transiently induced within a few days, simultaneously with MGP, followed by a subsequent and sustained induction of BMP-2 that lasted for over a week. However, neither BMP-4 nor BMP-2 was produced by BM stromal cells when cultured with dissociated BM hematopoietic cells using the transwell apparatus. Therefore, the induction of BMP2/BMP4 was dependent on physical association of hematopoietic cells and stromal cells. GST-pulldown assays and mammalian two-hybrid assays confirmed that MGP specifically interacted with both BMP-4 and BMP-2. To elucidate the mechanism of action of the blocking antibody against MGP, we tested if the antibody inhibited the interaction between MGP and BMP-4. Indeed, serial GST pulldown assays disclosed that the addition of the antibody specifically abolished the interaction between GST-MGP and BMP-4. These results indicate that MGP might act as a niche factor for normal and malignant HSPCs, at least in part, through interacting with, and modulating the action of, BMP-4 and BMP-2. DisclosuresNo relevant conflicts of interest to declare.

Disruption of cardiac Med1 inhibits RNA polymerase II promoter occupancy and promotes chromatin remodeling.

The Mediator coactivator complex directs gene-specific expression by binding distal enhancer-bound transcription factors through its Med1 subunit while bridging to RNA polymerase II (Pol II) at gene promoters. In addition, Mediator scaffolds epigenetic modifying enzymes that determine local DNA accessibility. Previously, we found that deletion of Med1 in cardiomyocytes deregulates more than 5,000 genes and promotes acute heart failure. Therefore, we hypothesized that Med1 deficiency disrupts enhancer-promoter coupling. Using chromatin immunoprecipitation-coupled deep sequencing (ChIP-seq; n = 3/ChIP assay), we found that the Pol II pausing index is increased in Med1 knockout versus floxed control mouse hearts primarily due to a decrease in Pol II occupancy at the majority of transcriptional start sites without a corresponding increase in elongating species. Parallel ChIP-seq assays reveal that Med1-dependent gene expression correlates strongly with histone H3 K27 acetylation, which is indicative of open and active chromatin at transcriptional start sites, whereas H3 K27 trimethylated levels, representing condensed and repressed DNA, are broadly increased and inversely correlate with absolute expression levels. Furthermore, Med1 deletion leads to dynamic changes in acetyl-K27 associated superenhancer regions and their enriched transcription factor-binding motifs that are consistent with altered gene expression. Our findings suggest that Med1 is important in establishing enhancer-promoter coupling in the heart and supports the proposed role of Mediator in establishing preinitiation complex formation. We also found that Med1 determines chromatin accessibility within genes and enhancer regions and propose that the composition of transcription factors associated with superenhancer changes to direct gene-specific expression. NEW & NOTEWORTHY Based on our previous findings that transcriptional homeostasis and cardiac function are disturbed by cardiomyocyte deletion of the Mediator coactivator Med1 subunit, we investigated potential underlying changes in RNA polymerase II localization and global chromatin accessibility. Using chromatin immunoprecipitation sequencing, we found that disrupted transcription arises from a deficit in RNA polymerase II recruitment to gene promoters. Furthermore, active versus repressive chromatin marks are redistributed within gene loci and at enhancer regions correlated with gene expression changes.

WHIRLY1 Occupancy Affects Histone Lysine Modification and WRKY53 Transcription in Arabidopsis Developmental Manner.

Single-stranded DNA-binding proteins (SSBs) are assumed to involve in DNA replication, DNA repairmen, and gene transcription. Here, we provide the direct evidence on the functionality of an Arabidopsis SSB, WHIRLY1, by using loss- or gain-of-function lines. We show that WHIRLY1 binding to the promoter of WRKY53 represses the enrichment of H3K4me3, but enhances the enrichment of H3K9ac at the region contained WHIRLY1-binding sequences and TATA box or the translation start region of WRKY53, coincided with a recruitment of RNAPII. In vitro ChIP assays confirm that WHIRLY1 inhibits H3K4me3 enrichment at the preinitiation complex formation stage, while promotes H3K9ac enrichment and RNAPII recruitment at the elongation stage, consequently affecting the transcription of WRKY53. These results further explore the molecular actions underlying SSB-mediated gene transcription through epigenetic regulation in plant senescence.

Yeast Ded1 promotes 48S translation pre-initiation complex assembly in an mRNA-specific and eIF4F-dependent manner.

DEAD-box RNA helicase Ded1 is thought to resolve secondary structures in mRNA 5'-untranslated regions (5'-UTRs) that impede 48S preinitiation complex (PIC) formation at the initiation codon. We reconstituted Ded1 acceleration of 48S PIC assembly on native mRNAs in a pure system, and recapitulated increased Ded1-dependence of mRNAs that are Ded1-hyperdependent in vivo. Stem-loop (SL) structures in 5'-UTRs of native and synthetic mRNAs increased the Ded1 requirement to overcome their intrinsically low rates of 48S PIC recruitment. Ded1 acceleration of 48S assembly was greater in the presence of eIF4F, and domains mediating one or more Ded1 interactions with eIF4G or helicase eIF4A were required for efficient recruitment of all mRNAs; however, the relative importance of particular Ded1 and eIF4G domains were distinct for each mRNA. Our results account for the Ded1 hyper-dependence of mRNAs with structure-prone 5'-UTRs, and implicate an eIF4E·eIF4G·eIF4A·Ded1 complex in accelerating 48S PIC assembly on native mRNAs.

XPC is an RNA polymerase II cofactor recruiting ATAC to promoters by interacting with E2F1

The DNA damage sensor XPC is involved in nucleotide excision repair. Here we show that in the absence of damage, XPC co-localizes with RNA polymerase II (Pol II) and active post-translational histone modifications marks on a subset of class II promoters in human fibroblasts. XPC depletion triggers specific gene down-expression due to a drop in the deposition of histone H3K9 acetylation mark and pre-initiation complex formation. XPC interacts with the histone acetyltransferase KAT2A and specifically triggers the recruitment of the KAT2A-containing ATAC complex to the promoters of down-expressed genes. We show that a strong E2F1 signature characterizes the XPC/KAT2A-bound promoters and that XPC interacts with E2F1 and promotes its binding to its DNA element. Our data reveal that the DNA repair factor XPC is also an RNA polymerase II cofactor recruiting the ATAC coactivator complex to promoters by interacting with the DNA binding transcription factor E2F1.

Homozygous TAF8 mutation in a patient with intellectual disability results in undetectable TAF8 protein, but preserved RNA polymerase II transcription.

The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. In a child with intellectual disability, mild microcephaly, corpus callosum agenesis and poor growth, we identified a homozygous splice-site mutation in TAF8 (NM_138572.2: c.781–1G > A). Our data indicate that the patient’s mutation generates a frame shift and an unstable TAF8 mutant protein with an unrelated C-terminus. The mutant TAF8 protein could not be detected in extracts from the patient’s fibroblasts, indicating a loss of TAF8 function and that the mutation is most likely causative. Moreover, our immunoprecipitation and proteomic analyses show that in patient cells only partial TAF complexes exist and that the formation of the canonical TFIID is impaired. In contrast, loss of TAF8 in mouse embryonic stem cells and blastocysts leads to cell death and to a global decrease in Pol II transcription. Astonishingly however, in human TAF8 patient cells, we could not detect any cellular phenotype, significant changes in genome-wide Pol II occupancy and pre-mRNA transcription. Thus, the disorganization of the essential holo-TFIID complex did not affect global Pol II transcription in the patient’s fibroblasts. Our observations further suggest that partial TAF complexes, and/or an altered TFIID containing a mutated TAF8, could support human development and thus, the absence of holo-TFIID is less deleterious for transcription than originally predicted.

Mechanistic interrogation of the entry‐ and exit‐channel arms of eIF3

Eukaryotic translation initiation factor 3 (eIF3) is a pivotal player in the recruitment of mRNA to the translation pre‐initiation complex (PIC). Biochemical and genetic studies have identified interactions of eIF3 with the PIC near the mRNA entry and exit channels of the ribosome; structural studies have revealed that eIF3 binds the solvent face of the small (40S) ribosomal subunit, projecting arms into both mRNA channels and ultimately encircling the PIC. We recently probed the effects of a library of S. cerevisiae eIF3 functional variants using an in‐vitro reconstituted system. These experiments demonstrated that mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment of a native yeast mRNA. Using a series of model mRNAs to control the amount of nucleotide sequence in either the mRNA entry or exit channel, we further revealed a critical role for eIF3 in stabilizing mRNA at the exit channel. Together, these experiments are consistent with the entry‐ and exit‐channel arms of eIF3 playing distinct roles during PIC formation, mRNA recruitment, and scanning. We have also investigated the collaboration of eIF3 with Rps3/uS3 in stabilizing mRNA within the mRNA entry channel. We found that Rps3/uS3 residues previously implicated in the helicase activity of the elongating ribosome stabilize mRNA binding within ribosomal entry channel and enhance the effects of eIF3 on mRNA binding in both channels. More recently, we have observed that eIF3i and eIF3g contribute to the PIC‐dependent enhancement of eIF4A ATPase activity, which recent work suggests may function to remodel the mRNA entry channel. Our current work is focused on dissecting the interactions between eIF3, the PIC, and other initiation factors in both the mRNA entry and exit channels, as well as on the inter‐subunit face of the PIC, using both in‐vitro and next‐generation sequencing approaches.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The role of yeast RNA polymerase I initiation factor Core Factor subunit Rrn7 in promoter open complex formation

A central component in S. cerevisiae RNA polymerase I (Pol I) pre‐initiation complex (PIC) formation and transcriptional regulation is the evolutionarily conserved initiation factor termed Core Factor (CF). CF is a heterotrimer composed of Rrn6, Rrn11, and the TFIIB‐related factor Rrn7. Recent Cryo‐EM structures of the Pol I initiation complex reveal that CF resembles a right hand grasping promoter DNA in which key contacts are mediated by Rrn11 and Rrn7 (Han et al., eLife, 2017). Similar to its Pol II counterpart TFIIB, CF subunit Rrn7 consists of an N‐terminal zinc ribbon (ZR) domain followed by a linker region and two cyclin fold repeats. We systematically mutagenized the Rrn7 ZR and linker domains to further understand their role in Pol I transcription initiation. We found that ZR and linker mutants are still able to form the CF complex and assemble a PIC, yet are defective in promoter opening. These findings, paired with the Cryo‐EM structure, suggest that CF acts as a ratchet to drive promoter DNA into the active site of Pol I and that the Rrn7 ZR and linker domains play a dynamic role in ATP‐independent promoter opening.Support or Funding InformationThis work was supported by grants awarded to BAK: National Cancer Institute (5K22CA184235), Alexandrine and Alexander L. Sinsheimer Scholar award, and SUNY Research Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reconstitution of RNA Polymerase I Upstream Activating Factor and the Roles of Histones H3 and H4 in Complex Assembly

RNA polymerase I (Pol I) transcription in Saccharomyces cerevisiae requires four separate factors that recruit Pol I to the promoter to form a pre-initiation complex. Upstream Activating Factor (UAF) is one of two multi-subunit complexes that regulate pre-initiation complex formation by binding to the ribosomal DNA promoter and by stimulating recruitment of downstream Pol I factors. UAF is composed of Rrn9, Rrn5, Rrn10, Uaf30, and histones H3 and H4. We developed a recombinant Escherichia coli-based system to coexpress and purify transcriptionally active UAF complex and to investigate the importance of each subunit in complex formation. We found that no single subunit is required for UAF assembly, including histones H3 and H4. We also demonstrate that histone H3 is able to interact with each UAF-specific subunit, and show that there are at least two copies of histone H3 and one copy of H4 present in the complex. Together, our results provide a new model suggesting that UAF contains a hybrid H3–H4 tetramer-like subcomplex.

Two Distinct Regulatory Mechanisms of Transcriptional Initiation in Response to Nutrient Signaling.

SAGA (Spt-Ada-Gcn5-Acetyltransferase) and TFIID (transcription factor IID) have been previously shown to facilitate the formation of the PIC (pre-initiation complex) at the promoters of two distinct sets of genes. Here, we demonstrate that TFIID and SAGA differentially participate in the stimulation of PIC formation (and hence transcriptional initiation) at the promoter of PHO84, a gene for the high-affinity inorganic phosphate (Pi) transporter for crucial cellular functions, in response to nutrient signaling. We show that transcriptional initiation of PHO84 occurs predominantly in a TFIID-dependent manner in the absence of Pi in the growth medium. Such TFIID dependency is mediated via the NuA4 (nucleosome acetyltransferase of H4) histone acetyltransferase (HAT). Intriguingly, transcriptional initiation of PHO84 also occurs in the presence of Pi in the growth medium, predominantly via the SAGA complex, but independently of NuA4 HAT. Thus, Pi in the growth medium switches transcriptional initiation of PHO84 from NuA4-TFIID to SAGA dependency. Further, we find that both NuA4-TFIID- and SAGA-dependent transcriptional initiations of PHO84 are facilitated by the 19S proteasome subcomplex or regulatory particle (RP) via enhanced recruitment of the coactivators SAGA and NuA4 HAT, which promote TFIID-independent and -dependent PIC formation for transcriptional initiation, respectively. NuA4 HAT does not regulate activator binding to PHO84, but rather facilitates PIC formation for transcriptional initiation in the absence of Pi in the growth medium. On the other hand, SAGA promotes activator recruitment to PHO84 for transcriptional initiation in the growth medium containing Pi. Collectively, our results demonstrate two distinct stimulatory pathways for PIC formation (and hence transcriptional initiation) at PHO84 by TFIID, SAGA, NuA4, and 19S RP in the presence and absence of an essential nutrient, Pi, in the growth media, thus providing new regulatory mechanisms of transcriptional initiation in response to nutrient signaling.

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

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

The mediator complex in genomic and non-genomic signaling in cancer

Mediator is a conserved, multi-subunit macromolecular machine divided structurally into head, middle, and tail modules, along with a transiently associating kinase module. Mediator functions as an integrator of transcriptional regulatory activity by interacting with DNA-bound transcription factors and with RNA polymerase II (Pol II) to both activate and repress gene expression. Mediator has been shown to affect multiple steps in transcription, including chromatin looping between enhancers and promoters, pre-initiation complex formation, transcriptional elongation, and mRNA splicing. Individual Mediator subunits participate in regulation of gene expression by the estrogen and androgen receptors and are altered in a number of endocrine cancers, including breast and prostate cancer. In addition to its role in genomic signaling, MED12 has been implicated in non-genomic signaling by interacting with and activating TGF-beta receptor 2 in the cytoplasm. Recent structural studies have revealed extensive inter-domain interactions and complex architecture of the Mediator-Pol II complex, suggesting that Mediator is capable of reorganizing its conformation and composition to fit cellular needs. We propose that alterations in Mediator subunit expression that occur in various cancers could impact the organization and function of Mediator, resulting in changes in gene expression that promote malignancy. A better understanding of the role of Mediator in cancer could reveal new approaches to the diagnosis and treatment of Mediator-dependent endocrine cancers, especially in settings of therapy resistance.

Structure of the transcription activator target Tra1 within the chromatin modifying complex SAGA

The transcription co-activator complex SAGA is recruited to gene promoters by sequence-specific transcriptional activators and by chromatin modifications to promote pre-initiation complex formation. The yeast Tra1 subunit is the major target of acidic activators such as Gal4, VP16, or Gcn4 but little is known about its structural organization. The 430 kDa Tra1 subunit and its human homolog the transformation/transcription domain-associated protein TRRAP are members of the phosphatidyl 3-kinase-related kinase (PIKK) family. Here, we present the cryo-EM structure of the entire SAGA complex where the major target of activator binding, the 430 kDa Tra1 protein, is resolved with an average resolution of 5.7 Å. The high content of alpha-helices in Tra1 enabled tracing of the majority of its main chain. Our results highlight the integration of Tra1 within the major epigenetic regulator SAGA.

TFIIH generates a six-base-pair open complex during RNAP II transcription initiation and start-site scanning

Eukaryotic mRNA transcription initiation is directed by the formation of the megaDalton-sized pre-initiation complex (PIC). After PIC formation, double-stranded DNA is unwound to form a single-stranded DNA bubble and the template strand is loaded into the polymerase active site. DNA opening is catalyzed by Ssl2(XPB), the dsDNA translocase subunit of the basal transcription factor TFIIH. In yeast, transcription initiation proceeds through a scanning phase where downstream DNA is searched for optimal start-sites. Here, to test models for initial DNA opening and start-site scanning, we measure the DNA bubble sizes generated by Saccharomyces cerevisiae PICs in real time using single-molecule magnetic tweezers. We show that ATP hydrolysis by Ssl2 opens a 6 base-pair (bp) bubble that grows to 13 bp in the presence of NTPs. These observations support a two-step model wherein ATP-dependent Ssl2 translocation leads to a 6 bp open complex which RNA polymerase II expands via NTP-dependent RNA transcription.

A heterochromatin-dependent transcription machinery drives piRNA expression

Nuclear small RNA pathways safeguard genome integrity by establishing transcription-repressing heterochromatin at transposable elements. This inevitably also targets the transposon-rich source loci of the small RNAs themselves. How small RNA source loci are efficiently transcribed while transposon promoters are potently silenced, is not understood. Here, we show that transcription of Drosophila piRNA clusters—small RNA source loci in animal gonads—is enforced through RNA Polymerase II pre-initiation complex formation within repressive heterochromatin. This is accomplished through the TFIIA-L paralog Moonshiner, which is recruited to piRNA clusters via the Heterochromatin Protein-1 variant Rhino. Moonshiner triggers transcription initiation within piRNA clusters by recruiting the TATA box-binding protein (TBP)-related factor TRF2, an animal TFIID core variant. Thus, transcription of heterochromatic small RNA source loci relies on direct recruitment of the core transcriptional machinery to DNA via histone marks rather than sequence motifs, a concept that we argue is a recurring theme in evolution.

Genome-wide Single-Molecule Footprinting Reveals High RNA Polymerase II Turnover at Paused Promoters

SummaryTranscription initiation entails chromatin opening followed by pre-initiation complex formation and RNA polymerase II recruitment. Subsequent polymerase elongation requires additional signals, resulting in increased residence time downstream of the start site, a phenomenon referred to as pausing. Here, we harnessed single-molecule footprinting to quantify distinct steps of initiation in vivo throughout the Drosophila genome. This identifies the impact of promoter structure on initiation dynamics in relation to nucleosomal occupancy. Additionally, perturbation of transcriptional initiation reveals an unexpectedly high turnover of polymerases at paused promoters—an observation confirmed at the level of nascent RNAs. These observations argue that absence of elongation is largely caused by premature termination rather than by stable polymerase stalling. In support of this non-processive model, we observe that induction of the paused heat shock promoter depends on continuous initiation. Our study provides a framework to quantify protein binding at single-molecule resolution and refines concepts of transcriptional pausing.

A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription.

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes.

Evidence that Mediator is essential for Pol II transcription, but is not a required component of the preinitiation complex in vivo.

The Mediator complex has been described as a general transcription factor, but it is unclear if it is essential for Pol II transcription and/or is a required component of the preinitiation complex (PIC) in vivo. Here, we show that depletion of individual subunits, even those essential for cell growth, causes a general but only modest decrease in transcription. In contrast, simultaneous depletion of all Mediator modules causes a drastic decrease in transcription. Depletion of head or middle subunits, but not tail subunits, causes a downstream shift in the Pol II occupancy profile, suggesting that Mediator at the core promoter inhibits promoter escape. Interestingly, a functional PIC and Pol II transcription can occur when Mediator is not detected at core promoters. These results provide strong evidence that Mediator is essential for Pol II transcription and stimulates PIC formation, but it is not a required component of the PIC in vivo.

Ligand-induced rapid skeletal muscle atrophy in HSA-Fv2E-PERK transgenic mice

BackgroundFormation of 43S and 48S preinitiation complexes plays an important role in muscle protein synthesis. There is no muscle-wasting mouse model caused by a repressed 43S preinitiation complex assembly.ObjectiveThe aim of the present study was to develop a convenient mouse model of skeletal muscle wasting with repressed 43S preinitiation complex assembly.Material and methodsA ligand-activatable PERK derivative Fv2E-PERK causes the phosphorylation of eukaryotic initiation factor 2α (eIF2α), which inhibits 43S preinitiation complex assembly. Thus, muscle atrophic phenotypes, intracellular signaling pathways, and intracellular free amino acid profiles were investigated in human skeletal muscle α-actin (HSA) promoter-driven Fv2E-PERK transgenic (Tg) mice.ResultsHSA-Fv2E-PERK Tg mice treated with the artificial dimerizer AP20187 phosphorylates eIF2α in skeletal muscles and leads to severe muscle atrophy within a few days of ligand injection. Muscle atrophy was accompanied by a counter regulatory activation of mTORC1 signaling. Moreover, intracellular free amino acid levels were distinctively altered in the skeletal muscles of HSA-Fv2E-PERK Tg mice.ConclusionsAs a novel model of muscle wasting, HSA-Fv2E-PERK Tg mice provide a convenient tool for studying the pathogenesis of muscle loss and for assessing putative therapeutics.