Regulation of hepatocyte cell cycle re-entry by RNA polymerase II-associated Gdown1.

Liver is the central organ responsible for whole-body metabolism, and its constituent hepatocytes are the major players that carry out liver functions. Although they are highly differentiated and rarely divide, hepatocytes re-enter the cell cycle following hepatic loss due to liver damage or injury. However, the exact molecular mechanisms underlying cell cycle re-entry remain undefined. Gdown1 is an RNA polymerase II (Pol II)-associated protein that has been linked to the function of the Mediator transcriptional coactivator complex. We recently found that Gdown1 ablation in mouse liver leads to down-regulation of highly expressed liver-specific genes and a concomitant cell cycle re-entry associated with the induction of cell cycle-related genes. Unexpectedly, in view of a previously documented inhibitory effect on transcription initiation by Pol II in vitro, we found that Gdown1 is associated with elongating Pol II on the highly expressed genes and that its ablation leads to a reduced Pol II occupancy that correlates with the reduced expression of these genes. Based on these observations, we discuss the in vitro and in vivo functions of Gdown1 and consider mechanisms by which the dysregulated Pol II recruitment associated with Gdown1 loss might induce quiescent cell re-entry into the cell cycle.

Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells.

Translation is an essential step in gene expression. In this study, we used an improved SunTag system to label nascent proteins and image translation of single messenger ribonucleoproteins (mRNPs) in human cells. Using a dedicated reporter RNA, we observe that translation of single mRNPs stochastically turns on and off while they diffuse through the cytoplasm. We further measure a ribosome density of 1.3 per kilobase and an elongation rate of 13-18 amino acids per second. Tagging the endogenous POLR2A gene revealed similar elongation rates and ribosomal densities and that nearly all messenger RNAs (mRNAs) are engaged in translation. Remarkably, tagging of the heavy chain of dynein 1 (DYNC1H1) shows this mRNA accumulates in foci containing three to seven RNA molecules. These foci are translation sites and thus represent specialized translation factories. We also observe that DYNC1H1 polysomes are actively transported by motors, which may deliver the mature protein at appropriate cellular locations. The SunTag should be broadly applicable to study translational regulation in live single cells.

Full-length autonomous transposable elements are preferentially targeted by expression-dependent forms of RNA-directed DNA methylation.

BACKGROUND: Chromatin modifications such as DNA methylation are targeted to transposable elements by small RNAs in a process termed RNA-directed DNA methylation (RdDM). In plants, canonical RdDM functions through RNA polymerase IV to reinforce pre-existing transposable element silencing. Recent investigations have identified a "non-canonical" form of RdDM dependent on RNA polymerase II expression to initiate and re-establish silencing of active transposable elements. This expression-dependent RdDM mechanism functions through RNAi degradation of transposable element mRNAs into small RNAs guided by the RNA-dependent RNA polymerase 6 (RDR6) protein and is therefore referred to as RDR6-RdDM. RESULTS: We performed whole-genome MethylC-seq in 20 mutants that distinguish RdDM mechanisms when transposable elements are either transcriptionally silent or active. We identified a new mechanism of expression-dependent RdDM, which functions through DICER-LIKE3 (DCL3) but bypasses the requirement of both RNA polymerase IV and RDR6 (termed DCL3-RdDM). We found that RNA polymerase II expression-dependent forms of RdDM function on over 20 % of transcribed transposable elements, including the majority of full-length elements with all of the domains required for autonomous transposition. Lastly, we find that RDR6-RdDM preferentially targets long transposable elements due to the specificity of primary small RNAs to cleave full-length mRNAs. CONCLUSIONS: Expression-dependent forms of RdDM function to critically target DNA methylation to full-length and transcriptionally active transposable elements, suggesting that these pathways are key to suppressing mobilization. This targeting specificity is initiated on the mRNA cleavage-level, yet manifested as chromatin-level silencing that in plants is epigenetically inherited from generation to generation.

Comprehensive analysis of promoter-proximal RNA polymerase II pausing across mammalian cell types.

BACKGROUND: For many genes, RNA polymerase II stably pauses before transitioning to productive elongation. Although polymerase II pausing has been shown to be a mechanism for regulating transcriptional activation, the extent to which it is involved in control of mammalian gene expression and its relationship to chromatin structure remain poorly understood. RESULTS: Here, we analyze 85 RNA polymerase II chromatin immunoprecipitation (ChIP)-sequencing experiments from 35 different murine and human samples, as well as related genome-wide datasets, to gain new insights into the relationship between polymerase II pausing and gene regulation. Across cell and tissue types, paused genes (pausing index > 2) comprise approximately 60 % of expressed genes and are repeatedly associated with specific biological functions. Paused genes also have lower cell-to-cell expression variability. Increased pausing has a non-linear effect on gene expression levels, with moderately paused genes being expressed more highly than other paused genes. The highest gene expression levels are often achieved through a novel pause-release mechanism driven by high polymerase II initiation. In three datasets examining the impact of extracellular signals, genes responsive to stimulus have slightly lower pausing index on average than non-responsive genes, and rapid gene activation is linked to conditional pause-release. Both chromatin structure and local sequence composition near the transcription start site influence pausing, with divergent features between mammals and Drosophila. Most notably, in mammals pausing is positively correlated with histone H2A.Z occupancy at promoters. CONCLUSIONS: Our results provide new insights into the contribution of RNA polymerase II pausing in mammalian gene regulation and chromatin structure.

Ccr4-not regulates RNA polymerase I transcription and couples nutrient signaling to the control of ribosomal RNA biogenesis.

Ribosomal RNA synthesis is controlled by nutrient signaling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. mTORC1 regulates ribosomal RNA expression by affecting RNA Polymerase I (Pol I)-dependent transcription of the ribosomal DNA (rDNA) but the mechanisms involved remain obscure. This study provides evidence that the Ccr4-Not complex, which regulates RNA Polymerase II (Pol II) transcription, also functions downstream of mTORC1 to control Pol I activity. Ccr4-Not localizes to the rDNA and physically associates with the Pol I holoenzyme while Ccr4-Not disruption perturbs rDNA binding of multiple Pol I transcriptional regulators including core factor, the high mobility group protein Hmo1, and the SSU processome. Under nutrient rich conditions, Ccr4-Not suppresses Pol I initiation by regulating interactions with the essential transcription factor Rrn3. Additionally, Ccr4-Not disruption prevents reduced Pol I transcription when mTORC1 is inhibited suggesting Ccr4-Not bridges mTORC1 signaling with Pol I regulation. Analysis of the non-essential Pol I subunits demonstrated that the A34.5 subunit promotes, while the A12.2 and A14 subunits repress, Ccr4-Not interactions with Pol I. Furthermore, ccr4Δ is synthetically sick when paired with rpa12Δ and the double mutant has enhanced sensitivity to transcription elongation inhibition suggesting that Ccr4-Not functions to promote Pol I elongation. Intriguingly, while low concentrations of mTORC1 inhibitors completely inhibit growth of ccr4Δ, a ccr4Δ rpa12Δ rescues this growth defect suggesting that the sensitivity of Ccr4-Not mutants to mTORC1 inhibition is at least partially due to Pol I deregulation. Collectively, these data demonstrate a novel role for Ccr4-Not in Pol I transcriptional regulation that is required for bridging mTORC1 signaling to ribosomal RNA synthesis.

Depletion of the RNA-binding protein RBP33 results in increased expression of silenced RNA polymerase II transcripts in Trypanosoma brucei.

We have characterized the RNA-binding protein RBP33 in Trypanosoma brucei, and found that it localizes to the nucleus and is essential for viability. The subset of RNAs bound to RBP33 was determined by immunoprecipitation of ribonucleoprotein complexes followed by deep sequencing. Most RBP33-bound transcripts are predicted to be non-coding. Among these, over one-third are located close to the end of transcriptional units (TUs) or have an antisense orientation within a TU. Depletion of RBP33 resulted in an increase in the level of RNAs derived from regions that are normally silenced, such as strand-switch regions, retroposon and repeat sequences. Our work provides the first example of an RNA-binding protein involved in the regulation of gene silencing in trypanosomes.

Rpb3 promotes hepatocellular carcinoma through its N-terminus.

The expression of RNA polymerase II subunit 3 (Rpb3) was found frequent up-regulation in Hepatocellular carcinoma (HCC) tumors. Significant associations could also be drawn between increased expressions of Rpb3 and advance HCC staging and shorter disease-free survival of patients. Overexpression of Rpb3 increased HCC cell proliferation, migratory rate and tumor growth in nude mice, whereas suppression of Rpb3 using shRNA inhibited these effects. For mechanism study, we found that Rpb3 bound directly to Snail, downregulated E-cadherin, induced HCC cells epithelial-mesenchymal transition (EMT). In particular, N-terminus of Rpb3 blocked Rpb3 binding to Snail, inhibited Rpb3-high-expression HCC cells proliferation, migration, tumor growth in nude mice, and also inhibited DEN-induced liver tumorigenesis. Furthermore, N-terminus of Rpb3 did not inhibit normal liver cells or Rpb3-low-expression HCC cells proliferation. These findings suggest that N-terminus of Rpb3 selectively inhibits Rpb3-high-expression HCC cells proliferation. N-terminus of Rpb3 may be useful in treating patients diagnosed with Rpb3-high-expression HCC.

Negative elongation factor is required for the maintenance of proviral latency but does not induce promoter-proximal pausing of RNA polymerase II on the HIV long terminal repeat.

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.

The Mediator complex and transcription regulation.

The Mediator complex is a multi-subunit assembly that appears to be required for regulating expression of most RNA polymerase II (pol II) transcripts, which include protein-coding and most non-coding RNA genes. Mediator and pol II function within the pre-initiation complex (PIC), which consists of Mediator, pol II, TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH and is approximately 4.0 MDa in size. Mediator serves as a central scaffold within the PIC and helps regulate pol II activity in ways that remain poorly understood. Mediator is also generally targeted by sequence-specific, DNA-binding transcription factors (TFs) that work to control gene expression programs in response to developmental or environmental cues. At a basic level, Mediator functions by relaying signals from TFs directly to the pol II enzyme, thereby facilitating TF-dependent regulation of gene expression. Thus, Mediator is essential for converting biological inputs (communicated by TFs) to physiological responses (via changes in gene expression). In this review, we summarize an expansive body of research on the Mediator complex, with an emphasis on yeast and mammalian complexes. We focus on the basics that underlie Mediator function, such as its structure and subunit composition, and describe its broad regulatory influence on gene expression, ranging from chromatin architecture to transcription initiation and elongation, to mRNA processing. We also describe factors that influence Mediator structure and activity, including TFs, non-coding RNAs and the CDK8 module.

The C-terminal domain of Rpb1 functions on other RNA polymerase II subunits.

The C-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II (RNApII), coordinates recruitment of RNA- and chromatin-modifying factors to transcription complexes. It is unclear whether the CTD communicates with the catalytic core region of Rpb1 and thus must be physically connected, or instead can function as an independent domain. To address this question, CTD was transferred to other RNApII subunits. Fusions to Rpb4 or Rpb6, two RNApII subunits located near the original position of CTD, support viability in a strain carrying a truncated Rpb1. In contrast, CTD fusion to Rpb9 on the other side of RNApII does not. Rpb4-CTD and Rpb6-CTD proteins are functional for phosphorylation and recruitment of various factors, albeit with some restrictions and minor defects. Normal CTD functions are not transferred to RNApI or RNApIII by Rbp6-CTD. These results show that, with some spatial constraints, CTD can function even when disconnected from Rpb1.

Molecular characterization of a gene POLR2H encoded an essential subunit for RNA polymerase II from the Giant Panda (Ailuropoda Melanoleuca).

The Giant Panda is an endangered and valuable gene pool in genetic, its important functional gene POLR2H encodes an essential shared peptide H of RNA polymerases. The genomic DNA and cDNA sequences were cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) adopting touchdown-PCR and reverse transcription polymerase chain reaction (RT-PCR), respectively. The length of the genomic sequence of the Giant Panda is 3,285 bp, including five exons and four introns. The cDNA fragment cloned is 509 bp in length, containing an open reading frame of 453 bp encoding 150 amino acids. Alignment analysis indicated that both the cDNA and its deduced amino acid sequence were highly conserved. Protein structure prediction showed that there was one protein kinase C phosphorylation site, four casein kinase II phosphorylation sites and one amidation site in the POLR2H protein, further shaping advanced protein structure. The cDNA cloned was expressed in Escherichia coli, which indicated that POLR2H fusion with the N-terminally His-tagged form brought about the accumulation of an expected 20.5 kDa polypeptide in line with the predicted protein. On the basis of what has already been achieved in this study, further deep-in research will be conducted, which has great value in theory and practical significance.

PCBP1 is required for maintenance of the transcriptionally silent state in fully grown mouse oocytes.

Global transcriptional silencing in fully grown oocytes is a critical event during mammalian oogenesis. However, how this event is regulated remains elusive. Here, we provide evidence that poly(rC)-binding protein 1 (PCBP1), a protein found by us previously to be present in metaphase II (MII) mouse oocytes, participates in maintenance of the transcriptionally silent state in fully grown mouse oocytes. Knocking down Pcbp1 by microinjection of its specific siRNAs into fully grown germinal vesicle (GV) oocytes resulted in remarkable changes in their transcriptional state, including the disequilibrium between the number of oocytes with an NSN (non-surrounded nucleolus) and those with a SN (surrounded nucleolus), and obvious transcriptional reactiviation in oocytes with a SN configuration as evidenced by BrUTP incorporation assay and immunofluorescent labeling of phosphorylated RNA polymerase II CTD and trimethylated H3 lysine 4, markers for active transcription. Furthermore, in a comprehensive microarray analysis of the preovulatory oocyte transcriptome, an incredible number of nearly 4,000 transcripts were upregulated in the Pcbp1 knockdown groups. These data indicate that lack of the function of PCBP1 disrupts the quiescent status of transcription in the fully grown oocytes, and hence supporting a role of this protein in the regulation of global transcriptional silcencing in fully grown mouse oocytes.

Reference genes in the developing murine brain and in differentiating embryonic stem cells.

OBJECTIVES: Gene expression analysis via quantitative real-time PCR (qPCR) is a key approach in biological and medical research. Here, variations between runs and samples are compensated for by in-parallel analysis of reference genes, which require a most stable expression throughout all samples and experimental procedures to function as internal standards. In reality, there is no universal reference gene; but rather, assumed reference genes vary widely among various cell types. This demands an evaluation of reference genes for each specific experimental purpose, especially in the case of developmental studies. The aim of the present study was to identify suitable reference genes for gene expression analysis in the developing murine brain neocortex in vivo and in mouse embryonic stem cells (mESC) throughout differentiation in vitro. METHODS: The five candidate genes Actb, 18s, Gapdh, Hprt, and RpII were analyzed throughout development in vivo and in vitro using the quartiles of C(q) values, fold change, coefficient of variation (CV) and the difference between maximum minus twofold standard deviation and mean as the criteria to evaluate their expression stability. RESULTS: We found that RpII was the most stable expressed gene in mESC throughout differentiation, while in the developing murine neocortex Gapdh showed the highest expression stability. CONCLUSIONS: Based on our results, we suggest for gene expression analysis in the context of neurodevelopment the usage of RpII as a reference gene for mESC and Gapdh or Hprt for the murine neocortex.

Multiple exposures to drought 'train' transcriptional responses in Arabidopsis.

Pre-exposure to stress may alter plants' subsequent responses by producing faster and/or stronger reactions implying that plants exercise a form of 'stress memory'. The mechanisms of plants' stress memory responses are poorly understood leaving this fundamental biological question unanswered. Here we show that during recurring dehydration stresses Arabidopsis plants display transcriptional stress memory demonstrated by an increase in the rate of transcription and elevated transcript levels of a subset of the stress-response genes (trainable genes). During recovery (watered) states, trainable genes produce transcripts at basal (preinduced) levels, but remain associated with atypically high H3K4me3 and Ser5P polymerase II levels, indicating that RNA polymerase II is stalled. This is the first example of a stalled RNA polymerase II and its involvement in transcriptional memory in plants. These newly discovered phenomena might be a general feature of plant stress-response systems and could lead to novel approaches for increasing the flexibility of a plant's ability to respond to the environment.

Origins and activity of the Mediator complex.

The Mediator is a large, multisubunit RNA polymerase II transcriptional regulator that was first identified in Saccharomyces cerevisiae as a factor required for responsiveness of Pol II and the general initiation factors to DNA binding transactivators. Since its discovery in yeast, Mediator has been shown to be an integral and highly evolutionarily conserved component of the Pol II transcriptional machinery with critical roles in multiple stages of transcription, from regulation of assembly of the Pol II initiation complex to regulation of Pol II elongation. Here we provide a brief overview of the evolutionary origins of Mediator, its subunit composition, and its remarkably diverse collection of activities in Pol II transcription.

RNA polymerase II kinetics in polo polyadenylation signal selection.

Regulated alternative polyadenylation is an important feature of gene expression, but how gene transcription rate affects this process remains to be investigated. polo is a cell-cycle gene that uses two poly(A) signals in the 3' untranslated region (UTR) to produce alternative messenger RNAs that differ in their 3'UTR length. Using a mutant Drosophila strain that has a lower transcriptional elongation rate, we show that transcription kinetics can determine alternative poly(A) site selection. The physiological consequences of incorrect polo poly(A) site choice are of vital importance; transgenic flies lacking the distal poly(A) signal cannot produce the longer transcript and die at the pupa stage due to a failure in the proliferation of the precursor cells of the abdomen, the histoblasts. This is due to the low translation efficiency of the shorter transcript produced by proximal poly(A) site usage. Our results show that correct polo poly(A) site selection functions to provide the correct levels of protein expression necessary for histoblast proliferation, and that the kinetics of RNA polymerase II have an important role in the mechanism of alternative polyadenylation.

The protein interaction network of the human transcription machinery reveals a role for the conserved GTPase RPAP4/GPN1 and microtubule assembly in nuclear import and biogenesis of RNA polymerase II.

RNA polymerase II (RNAPII), the 12-subunit enzyme that synthesizes all mRNAs and several non-coding RNAs in eukaryotes, plays a central role in cell function. Although multiple proteins are known to regulate the activity of RNAPII during transcription, little is known about the machinery that controls the fate of the enzyme before or after transcription. We used systematic protein affinity purification coupled to mass spectrometry (AP-MS) to characterize the high resolution network of protein interactions of RNAPII in the soluble fraction of human cell extracts. Our analysis revealed that many components of this network participate in RNAPII biogenesis. We show here that RNAPII-associated protein 4 (RPAP4/GPN1) shuttles between the nucleus and the cytoplasm and regulates nuclear import of POLR2A/RPB1 and POLR2B/RPB2, the two largest subunits of RNAPII. RPAP4/GPN1 is a member of a newly discovered GTPase family that contains a unique and highly conserved GPN loop motif that we show is essential, in conjunction with its GTP-binding motifs, for nuclear localization of POLR2A/RPB1 in a process that also requires microtubule assembly. A model for RNAPII biogenesis is presented.

POLR2F, ATP6V0A1 and PRNP expression in colorectal cancer: new molecules with prognostic significance?

BACKGROUND: DNA-directed RNA polymerase II subunit F (POLR2F), a subunit of the V0 domain of the vacuolar ATPase (ATP6V0A1) and the prion protein (PRNP) are molecules of potential importance in carcinogenesis and targeted cancer therapy. However, their expression has not been studied in colorectal carcinomas. PATIENTS AND METHODS: Expression microarray data were analyzed using a novel computational tool to reveal elevated levels of POLR2F, ATP6V0A1 and PRNP in relapsed colorectal carcinoma patients. The mRNA levels of POLR2F, ATP6V0A1 and PRNP were evaluated by quantitative RT-PCR in 70 colorectal carcinomas and 17 normal tissue specimens and were correlated with clinicopathological parameters. RESULTS: POLR2F and PRNP were up-regulated in colorectal carcinomas. Moreover, a significant difference in the expression levels of all three molecules between the right colon and the rectum was observed. High expression levels of POLR2F and ATP6V0A1 correlated with improved 3-year survival. Moreover, PRNP expression constituted an independent prognostic factor of the 3-year survival in multivariate analysis. CONCLUSION: POLR2F and PRNP exhibited elevated levels in carcinomas compared to normal tissue samples suggesting a possible role for these molecules in colorectal cancer. The association of the three molecules with survival or disease prognosis warrants further investigation.

Genomic location of the human RNA polymerase II general machinery: evidence for a role of TFIIF and Rpb7 at both early and late stages of transcription.

The functions ascribed to the mammalian GTFs (general transcription factors) during the various stages of the RNAPII (RNA polymerase II) transcription reaction are based largely on in vitro studies. To gain insight as to the functions of the GTFs in living cells, we have analysed the genomic location of several human GTF and RNAPII subunits carrying a TAP (tandem-affinity purification) tag. ChIP (chromatin immunoprecipitation) experiments using anti-tag beads (TAP-ChIP) allowed the systematic localization of the tagged factors. Enrichment of regions located close to the TIS (transcriptional initiation site) versus further downstream TRs (transcribed regions) of nine human genes, selected for the minimal divergence of their alternative TIS, were analysed by QPCR (quantitative PCR). We show that, in contrast with reports using the yeast system, human TFIIF (transcription factor IIF) associates both with regions proximal to the TIS and with further downstream TRs, indicating an in vivo function in elongation for this GTF. Unexpectedly, we found that the Rpb7 subunit of RNAPII, known to be required only for the initiation phase of transcription, remains associated with the polymerase during early elongation. Moreover, ChIP experiments conducted under stress conditions suggest that Rpb7 is involved in the stabilization of transcribing polymerase molecules, from initiation to late elongation stages. Together, our results provide for the first time a general picture of GTF function during the RNAPII transcription reaction in live mammalian cells and show that TFIIF and Rpb7 are involved in both early and late transcriptional stages.

Regulation of HIV-1 latency by T-cell activation.

HIV-infected patients harbor approximately 10(5)-10(6) memory CD4 T-cells that contain fully integrated but transcriptionally silent HIV proviruses. While small in number, these latently infected cells form a drug-insensitive reservoir that importantly contributes to the life-long persistence of HIV despite highly effective antiviral therapy. In tissue culture, latent HIV proviruses can be activated when their cellular hosts are exposed to select proinflammatory cytokines or their T-cell receptors are ligated. However, due to a lack of potency and/or dose-limiting toxicity, attempts to purge virus from this latent reservoir in vivo with immune-activating agents, such as anti-CD3 antibodies and IL-2, have failed. A deeper understanding of the molecular underpinnings of HIV latency is clearly required, including determining whether viral latency is actively reinforced by transcriptional repressors, defining which inducible host transcription factors most effectively antagonize latency, and elucidating the role of chromatin in viral latency. Only through such an improved understanding will it be possible to identify combination therapies that might allow complete purging of the latent reservoir and to realize the difficult and elusive goal of complete eradication of HIV in infected patients.