Pol II Research Articles

SAFB restricts contact domain boundaries associated with L1 chimeric transcription

Long interspersed element-1 (LINE-1 or L1) comprises 17% of the human genome, continuously generates genetic variations, and causes disease in certain cases. However, the regulation and function of L1 remain poorly understood. Here, we uncover that L1 can enrich RNA polymerase IIs (RNA Pol IIs), express L1 chimeric transcripts, and create contact domain boundaries in human cells. This impact of L1 is restricted by a nuclear matrix protein scaffold attachment factor B (SAFB) that recognizes transcriptionally active L1s by binding L1 transcripts to inhibit RNA Pol II enrichment. Acute inhibition of RNA Pol II transcription abolishes the domain boundaries associated with L1 chimeric transcripts, indicating a transcription-dependent mechanism. Deleting L1 impairs domain boundary formation, and L1 insertions during evolution have introduced species-specific domain boundaries. Our data show that L1 can create RNA Pol II-enriched regions that alter genome organization and that SAFB regulates L1 and RNA Pol II activity to preserve gene regulation.

Coupling of the engineered DNA "mutator" to a biosensor as a new paradigm for activation of silent biosynthetic gene clusters in Streptomyces.

DNA replication fidelity in Streptomyces bacteria, prolific producers of many medically important secondary metabolites, is understudied, while in Escherichia coli it is controlled by DnaQ, the ϵ subunit of DNA polymerase III (DNA PolIII). Manipulation of dnaQ paralogues in Streptomyces lividans TK24, did not lead to increased spontaneous mutagenesis in this bacterium suggesting that S. lividans DNA PolIII uses an alternative exonuclease activity for proofreading. In Mycobacterium tuberculosis, such activity is attributed to the DnaE protein representing α subunit of DNA PolIII. Eight DnaE mutants designed based on the literature data were overexpressed in S. lividans, and recombinant strains overexpressing two of these mutants displayed markedly increased frequency of spontaneous mutagenesis (up to 1000-fold higher compared to the control). One of these ‘mutators’ was combined in S. lividans with a biosensor specific for antibiotic coelimycin, which biosynthetic gene cluster is present but not expressed in this strain. Colonies giving a positive biosensor signal appeared at a frequency of ca 10–5, and all of them were found to produce coelimycin congeners. This result confirmed that our approach can be applied for chemical- and radiation-free mutagenesis in Streptomyces leading to activation of orphan biosynthetic gene clusters and discovery of novel bioactive secondary metabolites.

Evidence of slightly increased Pol II pausing in UPF1-depleted Drosophila melanogaster cells.

Evidence of slightly increased Pol II pausing in UPF1-depleted Drosophila melanogaster cells.

Brf1 loss and not overexpression disrupts tissues homeostasis in the intestine, liver and pancreas

RNA polymerase III (Pol-III) transcribes tRNAs and other small RNAs essential for protein synthesis and cell growth. Pol-III is deregulated during carcinogenesis; however, its role in vivo has not been studied. To address this issue, we manipulated levels of Brf1, a Pol-III transcription factor that is essential for recruitment of Pol-III holoenzyme at tRNA genes in vivo. Knockout of Brf1 led to embryonic lethality at blastocyst stage. In contrast, heterozygous Brf1 mice were viable, fertile and of a normal size. Conditional deletion of Brf1 in gastrointestinal epithelial tissues, intestine, liver and pancreas, was incompatible with organ homeostasis. Deletion of Brf1 in adult intestine and liver induced apoptosis. However, Brf1 heterozygosity neither had gross effects in these epithelia nor did it modify tumorigenesis in the intestine or pancreas. Overexpression of BRF1 rescued the phenotypes of Brf1 deletion in intestine and liver but was unable to initiate tumorigenesis. Thus, Brf1 and Pol-III activity are absolutely essential for normal homeostasis during development and in adult epithelia. However, Brf1 overexpression or heterozygosity are unable to modify tumorigenesis, suggesting a permissive, but not driving role for Brf1 in the development of epithelial cancers of the pancreas and gut.

Rpb5, a subunit shared by eukaryotic RNA polymerases, cooperates with prefoldin-like Bud27/URI.

Rpb5 is one of the five common subunits to all eukaryotic RNA polymerases, which is conserved in archaea, but not in bacteria. Among these common subunits, it is the only one that is not interchangeable between yeasts and humans, and accounts for the functional incompatibility of yeast and human subunits. Rpb5 has been proposed to contribute to the gene-specific activation of RNA pol II, notably during the infectious cycle of the hepatitis B virus, and also to participate in general transcription mediated by all eukaryotic RNA pol. The structural analysis of Rpb5 and its interaction with different transcription factors, regulators and DNA, accounts for Rpb5 being necessary to maintain the correct conformation of the shelf module of RNA pol II, which favors the proper organization of the transcription bubble and the clamp closure of the enzyme.In this work we provide details about subunit Rpb5's structure, conservation and the role it plays in transcription regulation by analyzing the different interactions with several factors, as well as its participation in the assembly of the three RNA pols, in cooperation with prefoldin-like Bud27/URI.

Effects of cyclin-dependent kinase 9 (CDK9) on lytic replication of Kaposi′s sarcoma-associated herpesvirus (KSHV)

Objective To investigate the role of cyclin-dependent kinase 9 (CDK9) in regulating lytic replication of Kaposi′s sarcoma-associated herpesvirus (KSHV). Methods Percentages of red fluorescent protein (RFP) positive cells were counted after treating and stimulating iSLK.219 cells with FIT-039, a CDK9 specific inhibitor, and doxycycline (Dox), respectively, or transfecting and stimulating them with CDK9 siRNA (si-CDK9) and Dox, respectively. Quantitative real-time PCR was used to detect the expression of KSHV genes at mRNA level in TREx-K-Rta BCBL-1 cells treated with FIT-039 or transfected with si-CKD9 in the presence of Dox. Chromatin immunoprecipitation (ChIP) assay was used to examine the enrichment of RNA polymerase Ⅱ (RNA Pol Ⅱ) and phosphorylated CTD-S2 of RNA Pol Ⅱ on the PAN promoter in TREx-K-Rta BCBL-1 cells treated with FIT-039. Results Both FIT-039 intervention and CDK9 knockdown dramatically deceased the percentage of RFP-positive iSLK.219 cells and suppressed the expression of ORF50, PAN and K2 in TREx-K-Rta BCBL-1 cells at mRNA level. Furthermore, FIT-039 significantly inhibited the enrichment of RNA Pol Ⅱ and phosphorylated CTD-S2 of RNA Pol Ⅱ on the PAN promoter in TREx-K-Rta BCBL-1 cells. Conclusion CDK9 is involved in regulating the lytic replication of KSHV through promoting the phosphorylation of CTD-S2 of RNA Pol Ⅱ. Key words: Cyclin-dependent kinase 9; Kaposi′s sarcoma-associated herpesvirus; Lytic replication; Gene transcriptional elongation

The Epsilon Subunit of DNA Polymerase III in the Bacterial Response to Quinolones

Quinolones are widely prescribed, broad‐spectrum antibacterial drugs. Despite their widespread use, the molecular mechanisms of quinolone cytotoxicity are not clearly understood. Quinolones target topoisomerases, preventing the resealing of otherwise transient DNA breaks. Poisoned topoisomerase, or stabilized cleavage complexes, are not sufficient for cell death. The goal of this study is to determine how stabilized cleavage complexes are converted into irreversible double strand DNA breaks (DSBs) that accumulate and drive quinolone cytotoxicity.Previous studies identified an unexpected connection between dnaQ and quinolone‐induced DNA damage in Escherichia coli. The ɛ subunit of DNA polymerase III, encoded by dnaQ, is an exonuclease providing the 3′ ‐> 5′ proofreading activity of the core. The absence of ɛ causes higher mutation rates, slow growth, constitutive SOS, and a defective SOS response following quinolone treatment. We found that these phenotypes are separable, which implies that ɛ has multiple, separable roles, aside from proofreading.We explore the role of ɛ‐β interaction in quinolone‐induced DSB generation. Using two different types of cell lysis in conjunction with pulsed‐field gel electrophoresis, we distinguished between latent breaks at the cleavage complex and overt chromosomal breaks due to additional processing. We provide evidence that supports the replication run‐off model of DSB generation, whereby a stronger ɛ‐β interaction causes the replicase to physically interact with stabilized cleavage complexes rather than stalling or disassembling.Support or Funding InformationGettysburg College Research & Professional Development Grant & Howard Hughes Medical Institute ‐ Cross‐Disciplinary Sciences Institute at Gettysburg College

Role of retinoblastoma protein-associated protein 48 (RBBP4) in HIV-1 latency

Objective To investigate the role and mechanism of retinoblastoma protein-associated proteins 48 (RBBP4) in HIV-1 latency. Methods CEM-Bru cells latently infected with HIV-1 were stimulated with 25 ng/ml of tumor necrosis factor alpha (TNF-α) in combination with 10 ng/ml of interleukin-2 (IL-2). Chromatin immunoprecipitation (ChIP) was performed to detect the changes in RBBP4 and in histone deacetylases 1 and 2 (HDAC1/2) binding to long terminal repeat (LTR). Binding activities of HDAC1/2 and RNA polymerase Ⅱ (RNA Pol Ⅱ) to LTR and acetylated histone H3 at LTR region were detected by ChIP after partially interfering the expression of RBBP4 in CEM-Bru cells with electroporation. Initiating and elongated transcripts were measured by RT-PCR. Results The binding activities of RBBP4 and HDAC1/2 to LTR in HIV-1 latently infected cells were enhanced significantly as compared with those in TNF-α and IL-2 co-stimulated cells. Fewer RBBP4 and HDAC1/2 bound to LTR following the interference of RBBP4 expression, which was accompanied with enhanced histone acetylation and strengthened binding activity of RNA Pol Ⅱ to LTR. Moreover, more initiating transcripts were detected in HIV-1 latently infected cells after the RBBP4 expression was interfered by electroporation. Conclusion RBBP4 contributes to the maintenance of HIV-1 latency, in which HDAC1 and HDAC2 might be involved. Key words: RBBP4; HDAC1; HDAC2; HIV-1; Latency

The DNA polymerase III holoenzyme contains γ and is not a trimeric polymerase.

There is widespread agreement that the clamp loader of the Escherichia coli replicase has the composition DnaX3δδ’χψ. Two DnaX proteins exist in E. coli, full length τ and a truncated γ that is created by ribosomal frameshifting. τ binds DNA polymerase III tightly; γ does not. There is a controversy as to whether or not DNA polymerase III holoenzyme (Pol III HE) contains γ. A three-τ form of Pol III HE would contain three Pol IIIs. Proponents of the three-τ hypothesis have claimed that γ found in Pol III HE might be a proteolysis product of τ. To resolve this controversy, we constructed a strain that expressed only τ from a mutated chromosomal dnaX. γ containing a C-terminal biotinylation tag (γ-Ctag) was provided in trans at physiological levels from a plasmid. A 2000-fold purification of Pol III* (all Pol III HE subunits except β) from this strain contained one molecule of γ-Ctag per Pol III* assembly, indicating that the dominant form of Pol III* in cells is Pol III2τ2 γδδ’χψ. Revealing a role for γ in cells, mutants that express only τ display sensitivity to ultraviolet light and reduction in DNA Pol IV-dependent mutagenesis associated with double-strand-break repair, and impaired maintenance of an F’ episome.

Annotation of genomics data by the bidirectional hidden Markov model unveils variations in Pol II transcription cycle

Annotation of genomics data by the bidirectional hidden Markov model unveils variations in Pol II transcription cycle

Retraction. Polymerase exchange during Okazaki fragment synthesis observed in living cells.

DNA replication machineries have been studied extensively, but the kinetics of action of their components remains largely unknown. We report a study of DNA synthesis during replication in living Escherichia coli cells. Using single-molecule microscopy, we observed repetitive fluorescence bursts of single polymerase IIIs (Pol IIIs), indicating polymerase exchange at the replication fork. Fluctuations in the amount of DNA-bound single-stranded DNA-binding protein (SSB) reflect different speeds for the leading- and lagging-strand DNA polymerases. Coincidence analyses of Pol III and SSB fluctuations show that they correspond to the lagging-strand synthesis and suggest the use of a new Pol III for each Okazaki fragment. Based on exchanges involving two Pol IIIs, we propose that the third polymerase in the replisome is involved in lagging-strand synthesis.

Rescue of DNA damage-stalled RNA Pol II: histone H2B in action.

RNA Pol II elongation in eukaryotes is coupled with a series of histone modifications. Elongating RNA Pol II can be strongly stalled by lesions on the DNA template. However, it is unclear whether RNA Pol II stalling affects elongation-associated histone modifications. We have explored this important question by investigating the function of histone H2B mono-ubiquitylation (H2Bub), a well-characterized epigenetic mark associated with RNA Pol II elongation, in the cellular response to DNA lesions induced by ultraviolet (UV) radiation. We found that, in contrast to transcription elongation, RNA Pol II stalling induced by UV lesions triggers rapid and significant H2B deubiquitylation that removes ubiquitin from H2B. Interestingly, in yeast mutant cells that lack H2B deubiquitylation enzymes, rescue of the stalled RNA Pol II by transcription-coupled repair (TCR) is significantly impaired. Thus, our study has established a direct connection between RNA Pol II stalling and a histone modification response.

Characterization of family D DNA polymerase from Thermococcus sp. 9°N.

Accurate DNA replication is essential for maintenance of every genome. All archaeal genomes except Crenarchaea, encode for a member of Family B (polB) and Family D (polD) DNA polymerases. Gene deletion studies in Thermococcus kodakaraensis and Methanococcus maripaludis show that polD is the only essential DNA polymerase in these organisms. Thus, polD may be the primary replicative DNA polymerase for both leading and lagging strand synthesis. To understand this unique archaeal enzyme, we report the biochemical characterization of a heterodimeric polD from Thermococcus. PolD contains both DNA polymerase and proofreading 3′–5′ exonuclease activities to ensure efficient and accurate genome duplication. The polD incorporation fidelity was determined for the first time. Despite containing 3′–5′ exonuclease proofreading activity, polD has a relatively high error rate (95 × 10−5) compared to polB (19 × 10−5) and at least 10-fold higher than the polB DNA polymerases from yeast (polε and polδ) or Escherichia coli DNA polIII holoenzyme. The implications of polD fidelity and biochemical properties in leading and lagging strand synthesis are discussed.Electronic supplementary materialThe online version of this article (doi:10.1007/s00792-014-0646-9) contains supplementary material, which is available to authorized users.

The role of CDK9 in myogenesis

Cdk9 is a member of cyclin-dependent kinases (cdks). It is expressed in human and murine tissues with high levels in terminally differentiated cells. It is involved in the regulation of transcriptional elongation via phosphorylation of CTD of RNA polII (1). It has been demonstrated that CdK9, complexed with CyT2a, plays a role in the activation of myogenic program. Moreover Cdk9-CyT2a activity is not down-regulated in myotube formation, but its activation contributes to the transcriptional activity MyoD-mediated during myogenic program (2). Recently, a 55 kDa protein called cdk9-55 has been identified. Cdk9-55 is significantly upregulated in cells induced to differentiate, either in C2C12 cells or in satellite isolated cells. In addition, it has been demonstrated that there is a clear induction of cdk9-55 expression in injured skeletal muscles (3). In order to deepen the discussion and extend the understanding of the biological role of the two isoforms of cdk9 during skeletal muscle differentiation process, we report the role of the two isoforms during in vivo mouse myogenesis. We have analyzed the behavior of CDk9-42 and CDK9-55 in primary and secondary myogenesis. Of particular interest are the apparent inverse correlation between the two cdk9 isoforms during the events of limbs formation and how the expression of two CDK9 isoforms, in combination with specific positive (Cycline T1,T2,k) or negative regulators (7SKsnRNA/Hexim1), is associated to a specific cellular phenotype and correlated to a specific function.

Computer and statistical analysis of transcription factor binding and chromatin modifications by ChIP-seq data in embryonic stem cell.

Advances in high throughput sequencing technology have enabled the identification of transcription factor (TF) binding sites in genome scale. TF binding studies are important for medical applications and stem cell research. Somatic cells can be reprogrammed to a pluripotent state by the combined introduction of factors such as Oct4, Sox2, c-Myc, Klf4. These reprogrammed cells share many characteristics with embryonic stem cells (ESCs) and are known as induced pluripotent stem cells (iPSCs). The signaling requirements for maintenance of human and murine embryonic stem cells (ESCs) differ considerably. Genome wide ChIP-seq TF binding maps in mouse stem cells include Oct4, Sox2, Nanog, Tbx3, Smad2 as well as group of other factors. ChIP-seq allows study of new candidate transcription factors for reprogramming. It was shown that Nr5a2 could replace Oct4 for reprogramming. Epigenetic modifications play important role in regulation of gene expression adding additional complexity to transcription network functioning. We have studied associations between different histone modification using published data together with RNA Pol II sites. We found strong associations between activation marks and TF binding sites and present it qualitatively. To meet issues of statistical analysis of genome ChIP-sequencing maps we developed computer program to filter out noise signals and find significant association between binding site affinity and number of sequence reads. The data provide new insights into the function of chromatin organization and regulation in stem cells.

Correlation between heparanase and vascular endothelial growth factor in human retinal microvascular endothelial cells induced by hypoxia

Objective To investigate the effects of heparanase and vascular endothelial growth factor (VEGF) and their correlation in CoCl2-induced human retinal microvascular endothelial cells (HRECs) in an hypoxia model.Methods Human eyes were selected to establish CoCl2-induced HRECs hypoxia model in this study.Four experimental groups were studied:normal control group,hypoxia group (CoCl2 100 μmol/L,48 hours),PI-88 group (specific competitive inhibitor of heparanase: phosphomannopentaose sulfate,PI-88,5 μg/ml,combined with CoCl2 100 μmol/L,48 hours) and PBS control group.Heparanase,VEGF and Pol Ⅱ expression in HRECs of normal and hypoxia group were analyzed with immunofluorescence.Western blot was used to evaluate the expression of heparanase and VEGF in HRECs of normal,hypoxia,PI-88 and PBS control groups. Results Immunofluorescence studies showed that the expression of heparanase and VEGF in cytoplasm was intense in hypoxia HRECs,but faint in normal group.Heparanase was also observed in the nucleus of hypoxia HRECs.Western blot results showed that the expression of Hpa and VEGF protein was increased significantly in hypoxia group compared with normal group (Hpa:F=- 4.005,P ＜ 0.05 ; VEGF:F =- 4.063,P ＜ 0.05),and VEGF was decreased in HRECs treated with PI-88 (F=5.963,P＜0.05).Conclusions Heparanase is up-regulated that resulted in increase of VEGF expression,therefore enhanced angiogenesis in CoCl2-induced hypoxia HRECs. Key words: Retinal neovascularization/pathophysiology; Vascular endothelial growth factors; Anoxia; Retinal vessels; Endothelial cells

RETRACTED: Polymerase exchange during Okazaki fragment synthesis observed in living cells.

DNA replication machineries have been studied extensively, but the kinetics of action of their components remains largely unknown. We report a study of DNA synthesis during replication in living Escherichia coli cells. Using single-molecule microscopy, we observed repetitive fluorescence bursts of single polymerase IIIs (Pol IIIs), indicating polymerase exchange at the replication fork. Fluctuations in the amount of DNA-bound single-stranded DNA-binding protein (SSB) reflect different speeds for the leading- and lagging-strand DNA polymerases. Coincidence analyses of Pol III and SSB fluctuations show that they correspond to the lagging-strand synthesis and suggest the use of a new Pol III for each Okazaki fragment. Based on exchanges involving two Pol IIIs, we propose that the third polymerase in the replisome is involved in lagging-strand synthesis.

Bacterial replicases and related polymerases

Bacterial replicases are complex, tripartite replicative machines. They contain a polymerase, Pol III, a β(2) processivity factor and a DnaX complex ATPase that loads β(2) onto DNA and chaperones Pol III onto the newly loaded β(2). Many bacteria encode both a full length τ and a shorter γ form of DnaX by a variety of mechanisms. The polymerase catalytic subunit of Pol III, α, contains a PHP domain that not only binds to prototypical ɛ Mg(2+)-dependent exonuclease, but also contains a second Zn(2+)-dependent proofreading exonuclease, at least in some bacteria. Replication of the chromosomes of low GC Gram-positive bacteria require two Pol IIIs, one of which, DnaE, appears to extend RNA primers a only short distance before handing the product off to the major replicase, PolC. Other bacteria encode a second Pol III (ImuC) that apparently replaces Pol V, required for induced mutagenesis in E. coli. Approaches that permit simultaneous biochemical screening of all components of complex bacterial replicases promise inhibitors of specific protein targets and reaction stages.

Pol III picks up the pieces

Pol III picks up the pieces

Genomic Analyses of Transcription Factor Binding, Histone Acetylation, and Gene Expression Reveal Mechanistically Distinct Classes of Estrogen-Regulated Promoters

To explore the global mechanisms of estrogen-regulated transcription, we used chromatin immunoprecipitation coupled with DNA microarrays to determine the localization of RNA polymerase II (Pol II), estrogen receptor alpha (ERalpha), steroid receptor coactivator proteins (SRC), and acetylated histones H3/H4 (AcH) at estrogen-regulated promoters in MCF-7 cells with or without estradiol (E2) treatment. In addition, we correlated factor occupancy with gene expression and the presence of transcription factor binding elements. Using this integrative approach, we defined a set of 58 direct E2 target genes based on E2-regulated Pol II occupancy and classified their promoters based on factor binding, histone modification, and transcriptional output. Many of these direct E2 target genes exhibit interesting modes of regulation and biological activities, some of which may be relevant to the onset and proliferation of breast cancers. Our studies indicate that about one-third of these direct E2 target genes contain promoter-proximal ERalpha-binding sites, which is considerably more than previous estimates. Some of these genes represent possible novel targets for regulation through the ERalpha/AP-1 tethering pathway. Our studies have also revealed several previously uncharacterized global features of E2-regulated gene expression, including strong positive correlations between Pol II occupancy and AcH levels, as well as between the E2-dependent recruitment of ERalpha and SRC at the promoters of E2-stimulated genes. Furthermore, our studies have revealed new mechanistic insights into E2-regulated gene expression, including the absence of SRC binding at E2-repressed genes and the presence of constitutively bound, promoter-proximally paused Pol IIs at some E2-regulated promoters. These mechanistic insights are likely to be relevant for understanding gene regulation by a wide variety of nuclear receptors.