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RNA Polymerase II (Pol II) transcriptional recycling is a mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. We describe an in vitro methodology facilitating unbiased identification of putative RNA Pol II transcriptional recycling factors and quantitative measurement of transcriptional output from recycled transcriptional components. Proof-of-principle experiments identified PAF1 complex components among recycling factors and detected defective transcriptional output from Pol II recycling following PAF1 depletion. Dynamic ChIP-seq confirmed PAF1 silencing triggered defective Pol II recycling in human cells. Prostate tumors exhibited enhanced transcriptional recycling, which was attenuated by antibody-based PAF1 depletion. These findings identify Pol II recycling as a potential target in cancer and demonstrate the applicability of in vitro and cellular transcription assays to characterize Pol II recycling in other disease states., RNA Polymerase II (Pol II) recycling can influence transcription efficiency. Here the authors describe an approach aimed at facilitating the identification of factors involved in Pol II recycling and identify PAF1 complex components as mediators of recycling.

Tau is modified with O-GlcNAcylation extensively in human brain. The O-GlcNAcylation levels of tau are decreased in Alzheimer's disease (AD) brain. Sirtuin type 1 (SIRT1) is an enzyme that deacetylates proteins including transcriptional factors and associates with neurodegenerative diseases, such as AD. Aberrant SIRT1 expression levels in AD brain is in parallel with the accumulation of tau. cAMP response element binding protein (CREB), a cellular transcription factor, plays a critical role in learning and memory. In this present study, we found SIRT1 deacetylates CREB and inhibits phosphorylation of CREB at Ser133. The inactivated CREB suppresses OGT expression and therefore decreases the O-GlcNAcylation of tau and thus increases the phosphorylation of tau at specific sites. These findings suggest that SIRT1 may be a potential therapeutic target for treating tauopathies.

RBM10 is an RNA-binding protein that regulates alternative splicing (AS). It localizes to the extra-nucleolar nucleoplasm and S1-1 nuclear bodies (NBs) in the nucleus. We investigated the biological significance of this localization in relation to its molecular function. Our analyses, employing deletion mutants, revealed that RBM10 possesses two S1-1 NB-targeting sequences (NBTSs), one in the KEKE motif region and another in the C2H2 Zn finger (ZnF). These NBTSs act synergistically to localize RBM10 to S1-1 NBs. The C2H2 ZnF not only acts as an NBTS, but is also essential for AS regulation by RBM10. Moreover, RBM10 does not participate in S1-1 NB formation, and without alterations of RBM10 protein levels, its NB-localization changes, increasing as cellular transcriptional activity declines, and vice versa. These results indicate that RBM10 is a transient component of S1-1 NBs and is sequestered in NBs via its NBTSs when cellular transcription decreases. We propose that the C2H2 ZnF exerts its NB-targeting activity when RBM10 is unbound by pre-mRNAs, and that NB-localization of RBM10 is a mechanism to control its AS activity in the nucleus.Note that the previous title of this manuscript was Targeting of RBM10 to S1-1 Nuclear Bodies: Targeting Sequences and its Biological Significance.

Introduction: The cell cycle cyclin-dependent kinases (CDKs) play a critical role in controlling the transition between cell cycle phases, as well as cellular transcription. Aberrant CDK activation...

Single-cell transcriptomics (SCT) is a tour de force in the era of big omics data that has led to the accumulation of massive cellular transcription data at an astounding resolution of single cells. It provides valuable insights into cells previously unachieved by bulk cell analysis and is proving crucial in uncovering cellular heterogeneity, identifying rare cell populations, distinct cell-lineage trajectories, and mechanisms involved in complex cellular processes. SCT data is highly complex and necessitates advanced statistical and computational methods for analysis. This review provides a comprehensive overview of the steps in a typical SCT workflow, starting from experimental protocol to data analysis, deliberating various pipelines used. We discuss recent trends, challenges, machine learning methods for data analysis, and future prospects. We conclude by listing the multitude of scRNA-seq data applications and how it shall revolutionize our understanding of cellular biology and diseases.

Cannabis use is frequent among people living with human immunodeficiency virus (HIV) and is associated with reduced systemic inflammation. We observed a faster HIV DNA decay during antiretroviral therapy among cannabis users, compared to those with no drug use. No cannabis effect was observed on cellular HIV RNA transcription.

Cannabis use is frequent among people living with HIV and is associated with reduced systemic inflammation. We observed a faster HIV DNA decay during antiretroviral therapy among cannabis users, compared to no drug use. No cannabis-effect was observed on cellular HIV RNA transcription.

Copyright © 2015 Barichievy, Naidoo and Mhlanga. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms., On October 28th 1943 Winston Churchill said "we shape our buildings, and afterward our buildings shape us" (Humes, 1994). Churchill was pondering how and when to rebuild the British House of Commons, which had been destroyed by enemy bombs on May 10th 1941. The old House had been small and insufficient to hold all its members, but was restored to its original form in 1950 in order to recapture the "convenience and dignity" that the building had shaped into its parliamentary members. The circular loop whereby buildings or dwellings are shaped and go on to shape those that reside in them is also true of pathogens and their hosts. As obligate parasites, pathogens need to alter their cellular host environments to ensure survival. Typically pathogens modify cellular transcription profiles and in doing so, the pathogen in turn is affected, thereby closing the loop. As key orchestrators of gene expression, non-coding RNAs provide a vast and extremely precise set of tools for pathogens to target in order to shape the cellular environment. This review will focus on host non-coding RNAs that are manipulated by the infamous intracellular pathogen, the human immunodeficiency virus (HIV). We will briefly describe both short and long host non-coding RNAs and discuss how HIV gains control of these factors to ensure widespread dissemination throughout the host as well as the establishment of lifelong, chronic infection.

Human papillomaviruses are the causative agent of cancers in stratified epithelial surfaces. They replicate in the upper parts of the epithelium, where cells would normally be dying to produce a cornified layer. Therefore, they need to inhibit or delay differentiation and stimulate cell cycle progression to create an environment conducive for replication of the viral genome. The alterations both in differentiation and in the cell cycle are achieved by the viral proteins E6 and E7, which modulate cellular transcription mainly through their effects on p53 and the retinoblastoma family.

The varicella zoster virus (VZV) IE62 protein is involved in the activation of expression of all three kinetic classes of VZV proteins. Analysis of the viral promoter for VZV glycoprotein I has shown that the cellular factor Sp1 is involved in or required for the observed IE62 mediated activation. Co-immunoprecipitation experiments show that the two proteins are present in a complex in VZV-infected cells. Protein affinity pull-down assays using recombinant proteins showed that IE62 and Sp1 interact in the absence of any other viral and cellular proteins. Mapping studies using GST-fusion proteins containing truncations of IE62 and Sp1 have delimited the interacting regions to amino acids 612-778 in Sp1 and amino acids 226-299 in IE62. The region identified in Sp1 is involved in DNA-binding, synergistic Sp1 activation, and Sp1 interaction with cellular transcription factors. The interacting region identified in IE62 overlaps with or borders on sites involved in interactions with the VZV IE4 protein and the cellular factors TBP and TFIIB. Assays using wild-type and mutant promoter elements indicate that Sp1 is involved in recruitment of IE62 to the gI promoter and IE62 enhances Sp1 and TBP binding.

The DNA architectural proteins FIS and CRP are global regulators of transcription in Escherichia coli involved in the adjustment of cellular metabolism to varying growth conditions. We have previously demonstrated that FIS modulates the expression of the crp gene by functioning as its transcriptional repressor. Here we show that in turn, CRP is required to maintain the growth phase pattern of fis expression. We demonstrate the existence of a divergent promoter in the fis regulatory region, which reduces transcription of the fis promoter. In the absence of FIS, CRP activates fis transcription, thereby displacing the polymerase from the divergent promoter, whereas together FIS and CRP synergistically repress fis gene expression. These results provide evidence for a direct cross-talk between global regulators of cellular transcription during the growth phase. This cross-talk is manifested in alternate formation of functional nucleoprotein complexes exerting either activating or repressing effects on transcription.

p300 and CBP are two related transcriptional co-activator proteins required by many cellular transcription factors for activity. The adenovirus E1A protein binds p300 and CBP through its amino-terminus and conserved region (CR) 1. Fusing CR1 to a heterologous DNA-binding domain creates a potent transcriptional activator, suggesting that CR1 might activate transcription by recruiting p300/CBP to the promoter. We show that both p300 and CBP enhances CR1-dependent transactivation. However, this enhancement occurs independently of a direct interaction with E1A and does not correlate with the CR1 activator function.

Human papillomaviruses (HPVs) modulate expression of host microRNAs. Our deep-sequencing analysis of organotypic raft cultures identified microRNA 145 (miR-145) as a differentiation-dependent microRNA that has functionally active target sequences in the HPV-31 E1 and E2 open reading frames. Overexpression of miR-145 in HPV-positive cells resulted in reduced genome amplification and late gene expression, along with decreased levels of cellular transcription factor KLF-4. Our studies show that HPV modulates miR-145 expression to control its own life cycle.

A number of studies have shown that exposures to environmental magnetic fields (MFs) increase cellular transcription and enhance DNA synthesis. However, little is known about the basic mechanisms underlying specific biological responses to MFs. We looked directly at the effect of MFs by using a cell-free rabbit globin cDNA synthesis system. cDNA synthesis reaction mixtures were placed in each of four exposure conditions and were tested simultaneously. Condition one was the control, with no exposure during either single-strand or double-strand synthesis (M-M-). The second condition was MF exposure only during single-strand synthesis (M+M-). The third condition was exposure only during double-strand synthesis (M-M+). The fourth condition was exposure during both single-strand and double-strand synthesis (M+M+). Results showed that cDNA synthesis was affected by 5-100 microT, 60 Hz MFs. Double-strand cDNA synthesis increased with MF exposure only during double-strand synthesis reactions (M-M+), and the greatest increase of double-strand cDNA synthesis was detected when MF exposure was at 10 microT, only during double-strand synthesis. Double-strand cDNA synthesis decreased when only single-strand synthesis reactions were exposed (M+M-; 100 microT). An increase of cDNA synthesis caused increased synthesis of rabbit globin cDNA and large-sized molecules. These results suggest that exposure to MF induced structural changes of synthesized cDNA, therefore altering the amount of cDNA. Our results show that environmental MFs can significantly alter cDNA synthesis in a cell-free system.

Measurement of the Brn-3a cellular transcription factor in cervical smears from women referred for colposcopy may improve the detection of underlying cervical neoplasia. In those women referred with persisting borderline or mildly dyskaryotic smears, those who had histologically proven underlying CIN II/III, had statistically significant higher Brn-3a levels than those with a similar smear but histologically shown to have HPV, CIN I, or no cervical abnormality. These results indicate that Brn-3a could play an important role in the near future in improving cervical cancer screening.

The realization that the cellular transcription factor E2F is a target for the action of the retinoblastoma tumor suppressor protein, as well as related regulatory proteins, and that this regulation is disrupted by oncogenic products of the DNA tumor viruses, has led to studies that have provided important insights into molecular mechanisms of cellular growth control.

Drosophila larvae reared inside a micro-metal box with an internal field strength 0.004 microT, were treated with a magnetic field of 50 Hz, 8 microT. for 20 min. Control experienced 0.004 microT. Cellular transcript levels were assessed using slot blots and quantified using a Phosphorimager. Blots were hybridised using probes against HSP 70a, Histone 1.9, and Copia. The low frequency EMFs very significantly decreased transcript levels, indicating that experimental responses may be influenced by previous exposure or lack of previous exposure.

The evaluation of the anti-HIV-1 activity of synthetic lignanolides and their intermediates is reported. The antiviral activity was studied through luciferase-based assays targeting the HIV-1 promoter activation induced by either, the HIV-1 Tat protein or the cellular transcription factor NF-kappaB, both known as crucial factors in HIV-1 replication. Among the compounds tested, three of them 2, 4 and 13 were further analysed for their anti-HIV-1 activity by recombinant virus assays, showing a suitable profile for development of novel anti-HIV-1 drugs.

Serial analysis of gene expression, or SAGE, is an experimental technique designed to gain a direct and quantitative measure of gene expression. The SAGE method is based on the isolation of unique sequence tags (9-10 bp in length) from individual mRNAs and concatenation of tags serially into long DNA molecules for a lump-sum sequencing. The SAGE method can be applied to the studies exploring virtually any kinds of biological phenomena in which the changes in cellular transcription are responsible. SAGE is a highly competent technology that can not only give a global gene expression profile of a particular type of cell or tissue, but also help us identify a set of specific genes to the cellular conditions by comparing the profiles constructed for a pair of cells that are kept at different conditions. In this review, we present an outline of the original method, several studies achieved by using the method as a major strategic tool, technological difficulties and intrinsic problems that emerged, and improvements and modifications of the method to cope with these drawbacks. We then present our modified SAGE procedure that generates longer sequence tags (14 bp) rather in detail, and the profile (80K profile) derived from HeLa cells that is composed of 80000 tags obtained from a single library. In addition, a series of smaller profiles (2, 4, 10, 20 and 40K) was made by dividing the 80K profile. When we compared these smaller profiles with respect to tag counts for a number of genes, it became apparent that counts of most gene tags increase stably and constantly as the size of profiles increase, while several genes do not. This may be another problem we have to keep in mind, when the profiles are compared for the identification of 'specific genes'.

Previous genetic and biochemical analyses have indicated that the Epstein-Barr virus EBNA-2 amino terminus is important for primary B-lymphocyte growth transformation and may be involved in self-association. We now report that EBNA-2 has at least two domains, amino acids 1 to 60 and 96 to 210, which independently mediate homotypic association, 1 to 60 with 1 to 60 and 96 to 210 with 96 to 210. EBNA-2 self-association is likely to be critical to the ability of EBNA-2 to interact simultaneously with multiple cellular transcription factors, coactivators, and histone acetyltransferases through its RBPJκ binding and acidic activating domains.

Fruit flavor is a result of a complex mixture of numerous compounds. The formation of these compounds is closely correlated with the metabolic changes occurring during fruit maturation. Here, we describe the use of DNA microarrays and appropriate statistical analyses to dissect a complex developmental process. In doing so, we have identified a novel strawberry alcohol acyltransferase (SAAT) gene that plays a crucial role in flavor biogenesis in ripening fruit. Volatile esters are quantitatively and qualitatively the most important compounds providing fruity odors. Biochemical evidence for involvement of the SAAT gene in formation of fruity esters is provided by characterizing the recombinant protein expressed in Escherichia coli. The SAAT enzyme showed maximum activity with aliphatic medium-chain alcohols, whose corresponding esters are major components of strawberry volatiles. The enzyme was capable of utilizing short- and medium-chain, branched, and aromatic acyl-CoA molecules as cosubstrates. The results suggest that the formation of volatile esters in fruit is subject to the availability of acyl-CoA molecules and alcohol substrates and is dictated by the temporal expression pattern of the SAAT gene(s) and substrate specificity of the SAAT enzyme(s).

The adequate and on-time response of cellular transcription to internal needs and external stimuli is a delicate task. Is the transcriptional regulator p53 on call to permit rapid activation of its target genes? A new study reports interplay of p53 with a previously unidentified regulator, calcineurin-binding protein 1 (Cabin1), and provides evidence for such a mechanism.

Cellular transcription networks are conceptualized as distributed control systems that regulate gene expression.

Acquired immunodeficiency syndrome (AIDS) is a result of replication of the human immunodeficiency virus type 1 (HIV-1) predominantly in CD4+ T lymphocytes and macrophages. However, most of these cells in vivo are immunologically quiescent, a condition restricting HIV-1 replication. Vpr is an HIV-1 virion protein suspected to enhance HIV-1 replication in vivo. We demonstrate in this report that Vpr specifically activates HIV-1 long terminal repeat (LTR)-directed transcription. This effect is most pronounced on a minimal promoter from HIV-1 LTR containing the TATA box and binding motifs for the ubiquitous cellular transcription factor Sp1. Evidence is presented that Vpr interacts with Sp1 when Sp1 is bound to the Sp1 motifs within the HIV-1 LTR Both Vpr-Sp1 interaction and Vpr trans-activation require a central Leu/Ile-rich domain in Vpr. Our findings suggest that Vpr trans-activation through Sp1 is most critical for the immediate early transcription of HIV-1 when other positive regulators, such as NF-kappa B, are limited or inactive, a condition presumably present in vivo. By interacting with Sp1, Vpr also has the potential to influence cellular gene expression and cellular functions. Thus, therapeutic approaches directed toward blocking the Vpr trans-activation function could prove valuable in treating AIDS.

The mechanism(s) of pathogenesis caused by persistent viruses such as the human cytomegalovirus (HCMV) are strongly associated with the control of transcription. In this chapter we explore in vitro systems which suggest the interplay of both viral and cellular regulatory factors as major determinants in controlling the expression state of HCMV. Importantly, we address the issue of both cellular transcription factors and viral regulatory proteins as possible mediators in the pathogenesis associated with HCMV infection.

We have previously shown that the human immunodeficiency virus type 1 (HIV-1) Tat protein can activate a synthetic promoter containing consensus-binding sites for the cellular transcription factor Sp1. In this report, we show that a GAL-Tat fusion protein targeted via GAL4 DNA-binding sites can also trans activate an HIV-1 LTR promoter independently of the trans-activation response region. To show that the trans activation of the promoter by Tat directly involves the Sp1 protein, we have targeted a GAL-Sp1 fusion protein to the long terminal repeat promoter via upstream GAL4-binding sites. In the presence of Tat and GAL-Sp1, the promoter is synergistically trans activated at the transcriptional level, indicating that Tat and Sp1 functionally interact to trans activate the HIV-1 promoter. The Sp1 synergism is relatively specific, since another chimeric transcriptional activator, GAL-VP16, does not appear to be significantly synergistic with Tat.

Our study reports the occurrence of HIV-1 insertional activation events in vivo . Using a previously described PCR assay, small aliquots of broncho-alveolar lavage (BAL) cells obtained from AIDS patients were analyzed. Nine percent (5/54) of the aliquots contained proviral-host sequence transcripts indicating HIV-1 promoter insertion, whereas multiply spliced HIV-1 mRNAs were found in 28% (15/54) of the aliquots. In four of five events, insertions affect distinct cellular transcription units expressed in a T-cell line. To establish a ratio between provirus integration and promoter insertion events, an in vitro infection study was performed and transcripts containing HIV-1 and K- ras or CD4 gene sequences, respectively, were monitored. Given the randomness of retrovirus integration, 170 sense-oriented HIV integrations into these gene loci were predicted to occur. Three distinct promoter insertion events were observed, indicating that 1.8% of integrated proviruses transcribed adjacent genes. Based on this result and a mean of 257 proviral copies per 10 6 BAL cells, we would expect to observe 25 promoter insertion events in our in vivo study. That only five events were found may be due to the lower transcriptional activity of HIV-1 in vivo than that in cell cultures.

This paper describes recent work in this laboratory and others which indicates that DNA both in higher cells and in bacterial cells exists in a regularly condensed structure. Evidence from the study of purified DNA in the presence of mixtures of small molecules suggests that such mixtures confer a regular polyamine coating along the length of the DNA helix. The DNA-polyamine complex may be caused to condense by the hi stones in higher cells and by proteins as yet to be described in bacterial cells. Possibly these condensed structures are important in the regulation of cellular transcription.

The adenovirus E1A gene product trans-activates a number of viral and cellular promoters. The mechanism for this transcriptional induction was investigated with an in vivo exoIII mapping technique to assay for proteins that interact with an E1A-inducible promoter. A protein bound to the early E2 promoter was detected in wild-type infected cells. In the absence of E1A induction, specific interactions at the promoter could not be detected, as indicated by the absence of an exoIII-protected fragment. However, if conditions were established that allowed transcription of the E2 gene in the absence of E1A, the same exoIII protection was observed as was found in the presence of E1A. These results suggest a model in which the efficient utilization of the E2 promoter is mediated by a cellular transcription factor. In the absence of E1A, the interaction can take place, but slowly and inefficiently in comparison with the interaction in the presence of E1A.

Publisher Summary This chapter emphasizes the current picture of the regulation of adenoviral gene expression. The large body of work discussed in this chapter amply documents the complexity and intricacy of the regulation of adenoviral mRNA production. The most obvious generalization that can be made concerns the relative roles of transcriptional and posttranscriptional regulation. It seems obvious that transcriptional control must determine when during the productive cycle a particular transcription unit is expressed. However, transcription units that are transcribed in adenovirus-infected cells do not necessarily generate functional mRNA species, a situation exemplified by many cellular transcription units during the late phase of infection. Moreover, a subtle network of posttranscriptional regulatory circuits is superimposed upon transcriptional control mechanisms. These can determine both the nature of the end product generated from a given transcript and the relative concentrations of individual mRNA species that may be processed from that transcript. Thus, the fine tuning of regulation of adenoviral gene expression appears to be largely a posttranscriptional affair.