Showing papers on "Transcription (biology) published in 2012"

Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat

Abstract: Most of the mammalian genome is transcribed. This generates a vast repertoire of transcripts that includes protein-coding messenger RNAs, long non-coding RNAs (lncRNAs) and repetitive sequences, such as SINEs (short interspersed nuclear elements). A large percentage of ncRNAs are nuclear-enriched with unknown function. Antisense lncRNAs may form sense-antisense pairs by pairing with a protein-coding gene on the opposite strand to regulate epigenetic silencing, transcription and mRNA stability. Here we identify a nuclear-enriched lncRNA antisense to mouse ubiquitin carboxy-terminal hydrolase L1 (Uchl1), a gene involved in brain function and neurodegenerative diseases. Antisense Uchl1 increases UCHL1 protein synthesis at a post-transcriptional level, hereby identifying a new functional class of lncRNAs. Antisense Uchl1 activity depends on the presence of a 5' overlapping sequence and an embedded inverted SINEB2 element. These features are shared by other natural antisense transcripts and can confer regulatory activity to an artificial antisense to green fluorescent protein. Antisense Uchl1 function is under the control of stress signalling pathways, as mTORC1 inhibition by rapamycin causes an increase in UCHL1 protein that is associated to the shuttling of antisense Uchl1 RNA from the nucleus to the cytoplasm. Antisense Uchl1 RNA is then required for the association of the overlapping sense protein-coding mRNA to active polysomes for translation. These data reveal another layer of gene expression control at the post-transcriptional level.

Condition-Dependent Transcriptome Reveals High-Level Regulatory Architecture in Bacillus subtilis

Abstract: Bacteria adapt to environmental stimuli by adjusting their transcriptomes in a complex manner, the full potential of which has yet to be established for any individual bacterial species. Here, we report the transcriptomes of Bacillus subtilis exposed to a wide range of environmental and nutritional conditions that the organism might encounter in nature. We comprehensively mapped transcription units (TUs) and grouped 2935 promoters into regulons controlled by various RNA polymerase sigma factors, accounting for ~66% of the observed variance in transcriptional activity. This global classification of promoters and detailed description of TUs revealed that a large proportion of the detected antisense RNAs arose from potentially spurious transcription initiation by alternative sigma factors and from imperfect control of transcription termination.

Nucleic Acids and Molecular Biology

Abstract: A wide range of topics are covered, including articles on nucleic acid structure, through their interactions with proteins to the control of gene expressions. A number of authors address the subject of RNA, including the difficult but important subject of its chemical synthesis, the complexities of its structures and the mechanisms of transcript splicing. The probing of DNA structure is reviewed in papers on the applications of dydroxyl radical and 1,10 phenanthroline copper cleavages. A number of important DNA-protein interactions are discussed, including DNA polymerase, the tryptophan and deoR repressors, and the resolvase enzymes which cleave Holliday junctions in recombination. Gene transcription is also covered, from the points of view of DNA methylation, mammalian ribosomal and avian lysozyme genes, and the control of transcription in the proto-oncogene c-fos. Finally, the plant kingdom has not been forgotten with articles on development and transposition in plants.

The RNA polymerase II CTD coordinates transcription and RNA processing

Abstract: The C-terminal domain (CTD) of the RNA polymerase II largest subunit consists of multiple heptad repeats (consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), varying in number from 26 in yeast to 52 in vertebrates. The CTD functions to help couple transcription and processing of the nascent RNA and also plays roles in transcription elongation and termination. The CTD is subject to extensive post-translational modification, most notably phosphorylation, during the transcription cycle, which modulates its activities in the above processes. Therefore, understanding the nature of CTD modifications, including how they function and how they are regulated, is essential to understanding the mechanisms that control gene expression. While the significance of phosphorylation of Ser2 and Ser5 residues has been studied and appreciated for some time, several additional modifications have more recently been added to the CTD repertoire, and insight into their function has begun to emerge. Here, we review findings regarding modification and function of the CTD, highlighting the important role this unique domain plays in coordinating gene activity.

Genome-wide structure and organization of eukaryotic pre-initiation complexes

Abstract: Transcription and regulation of genes originate from transcription pre-initiation complexes (PICs). Their structural and positional organization across eukaryotic genomes is unknown. Here we applied lambda exonuclease to chromatin immunoprecipitates (termed ChIP-exo) to examine the precise location of 6,045 PICs in Saccharomyces. PICs, including RNA polymerase II and protein complexes TFIIA, TFIIB, TFIID (or TBP), TFIIE, TFIIF, TFIIH and TFIIK were positioned within promoters and excluded from coding regions. Exonuclease patterns were in agreement with crystallographic models of the PIC, and were sufficiently precise to identify TATA-like elements at so-called TATA-less promoters. These PICs and their transcription start sites were positionally constrained at TFIID-engaged downstream +1 nucleosomes. At TATA-box-containing promoters, which are depleted of TFIID, a +1 nucleosome was positioned to be in competition with the PIC, which may allow greater latitude in start-site selection. Our genomic localization of messenger RNA and non-coding RNA PICs reveals that two PICs, in inverted orientation, may occupy the flanking borders of nucleosome-free regions. Their unambiguous detection may help distinguish bona fide genes from transcriptional noise.

RNA polymerase II elongation control

Abstract: Regulation of the elongation phase of transcription by RNA polymerase II (Pol II) is utilized extensively to generate the pattern of mRNAs needed to specify cell types and to respond to environmental changes. After Pol II initiates, negative elongation factors cause it to pause in a promoter proximal position. These polymerases are poised to respond to the positive transcription elongation factor P-TEFb, and then enter productive elongation only under the appropriate set of signals to generate full-length properly processed mRNAs. Recent global analyses of Pol II and elongation factors, mechanisms that regulate P-TEFb involving the 7SK small nuclear ribonucleoprotein (snRNP), factors that control both the negative and positive elongation properties of Pol II, and the mRNA processing events that are coupled with elongation are discussed.

Airn Transcriptional Overlap, But Not Its lncRNA Products, Induces Imprinted Igf2r Silencing

Abstract: Mammalian imprinted genes often cluster with long noncoding (lnc) RNAs. Three lncRNAs that induce parental-specific silencing show hallmarks indicating that their transcription is more important than their product. To test whether Airn transcription or product silences the Igf2r gene, we shortened the endogenous lncRNA to different lengths. The results excluded a role for spliced and unspliced Airn lncRNA products and for Airn nuclear size and location in silencing Igf2r. Instead, silencing only required Airn transcriptional overlap of the Igf2r promoter, which interferes with RNA polymerase II recruitment in the absence of repressive chromatin. Such a repressor function for lncRNA transcriptional overlap reveals a gene silencing mechanism that may be widespread in the mammalian genome, given the abundance of lncRNA transcripts.

IFN-α inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome

Abstract: HBV infection remains a leading cause of death worldwide. IFN-α inhibits viral replication in vitro and in vivo, and pegylated IFN-α is a commonly administered treatment for individuals infected with HBV. The HBV genome contains a typical IFN-stimulated response element (ISRE), but the molecular mechanisms by which IFN-α suppresses HBV replication have not been established in relevant experimental systems. Here, we show that IFN-α inhibits HBV replication by decreasing the transcription of pregenomic RNA (pgRNA) and subgenomic RNA from the HBV covalently closed circular DNA (cccDNA) minichromosome, both in cultured cells in which HBV is replicating and in mice whose livers have been repopulated with human hepatocytes and infected with HBV. Administration of IFN-α resulted in cccDNA-bound histone hypoacetylation as well as active recruitment to the cccDNA of transcriptional corepressors. IFN-α treatment also reduced binding of the STAT1 and STAT2 transcription factors to active cccDNA. The inhibitory activity of IFN-α was linked to the IRSE, as IRSE-mutant HBV transcribed less pgRNA and could not be repressed by IFN-α treatment. Our results identify a molecular mechanism whereby IFN-α mediates epigenetic repression of HBV cccDNA transcriptional activity, which may assist in the development of novel effective therapeutics.

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

Abstract: 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.

HIV-1 reverse transcription

Abstract: Reverse transcription and integration are the defining features of the Retroviridae; the common name "retrovirus" derives from the fact that these viruses use a virally encoded enzyme, reverse transcriptase (RT), to convert their RNA genomes into DNA. Reverse transcription is an essential step in retroviral replication. This article presents an overview of reverse transcription, briefly describes the structure and function of RT, provides an introduction to some of the cellular and viral factors that can affect reverse transcription, and discusses fidelity and recombination, two processes in which reverse transcription plays an important role. In keeping with the theme of the collection, the emphasis is on HIV-1 and HIV-1 RT.

Transcriptional and Posttranscriptional Regulation of HIV-1 Gene Expression

Abstract: Control of HIV-1 gene expression depends on two viral regulatory proteins, Tat and Rev. Tat stimulates transcription elongation by directing the cellular transcriptional elongation factor P-TEFb to nascent RNA polymerases. Rev is required for the transport from the nucleus to the cytoplasm of the unspliced and incompletely spliced mRNAs that encode the structural proteins of the virus. Molecular studies of both proteins have revealed how they interact with the cellular machinery to control transcription from the viral LTR and regulate the levels of spliced and unspliced mRNAs. The regulatory feedback mechanisms driven by HIV-1 Tat and Rev ensure that HIV-1 transcription proceeds through distinct phases. In cells that are not fully activated, limiting levels of Tat and Rev act as potent blocks to premature virus production.

Genome-wide determination of RNA stability reveals hundreds of short-lived noncoding transcripts in mammals

Abstract: Whole transcriptome analyses using tiling microarrays (Bertone et al 2004) and deep sequencing (Nagalakshmi et al 2008) have revealed huge numbers of novel transcripts, including long and short noncoding RNAs (ncRNAs) The ratio of noncoding to protein-coding genomic regions increases as a function of developmental complexity (Mattick 2004), suggesting that revealing the functions of ncRNAs transcribed from noncoding genomic regions is important for understanding genome function in higher organisms The ncRNAs can be roughly classified into two groups: small transcripts, such as microRNAs and piwi-interacting RNAs (piRNAs), and long transcripts (Prasanth and Spector 2007) Although the biological importance of small ncRNAs has been documented in recent years, the physiological functions of long ncRNAs (lncRNAs) are poorly understood Recently, significant efforts have been applied to reveal the function of lncRNAs Several approaches have succeeded in identifying dozens of functional lncRNAs (Guttman et al 2009) However, the biological functions of the vast majority of lncRNAs remain unclear Thus, novel properties that can distinguish functional ncRNAs from transcriptional noise are required Numerous studies of mRNAs have revealed that changing the abundance of transcripts by regulated RNA degradation is a critical step in the control of various biological pathways (Keene 2010) It has been estimated that the mRNA abundance of 5%–10% of human genes is controlled through the regulation of RNA stability (Bolognani and Perrone-Bizzozero 2008) It has been proposed that the specific half-life of each mRNA is closely related to its physiological function (Lam et al 2001; Yang et al 2003; Raghavan and Bohjanen 2004; Sharova et al 2009; Rabani et al 2011; Schwanhausser et al 2011) Although mRNAs of most housekeeping genes have long half-lives, mRNAs of many regulatory genes, which encode proteins that are required for only a limited time in the cell—such as cell cycle regulators, factors responsible for responses to external stimuli, and regulators of growth or differentiation—often have short half-lives Moreover, most transcriptionally inducible genes are disproportionately classified into the group of genes with rapid mRNA turnover It is possible, therefore, that the RNA stability of noncoding transcripts also reflects their functions Traditionally, RNA decay has been assessed by blocking global transcription with transcriptional inhibitors, eg, actinomycin D (ActD), and subsequently monitoring ongoing RNA decay over time However, inhibitor-mediated global transcriptional arrest has a profoundly disruptive impact on cellular physiology and interferes with the precise determination of the RNA degradation rate (Blattner et al 2000; Friedel et al 2009) Here, we present a novel inhibitor-free method (5′-bromo-uridine immunoprecipitation chase, BRIC) that enables measurement of RNA decay under nondisruptive conditions Determination of the half-lives of whole transcripts by BRIC, combined with multifaceted deep sequencing (BRIC-seq), suggest that there is a relationship between the stability of ncRNAs, as well as mRNAs, and their physiological functions

An E. coli Cell-Free Expression Toolbox: Application to Synthetic Gene Circuits and Artificial Cells

Abstract: Cell-free protein synthesis is becoming a powerful technique to construct and to study complex informational processes in vitro. Engineering synthetic gene circuits in a test tube, however, is seriously limited by the transcription repertoire of modern cell-free systems, composed of only a few bacteriophage regulatory elements. Here, we report the construction and the phenomenological characterization of synthetic gene circuits engineered with a cell-free expression toolbox that works with the seven E. coli sigma factors. The E. coli endogenous holoenzyme E70 is used as the primary transcription machinery. Elementary circuit motifs, such as multiple stage cascades, AND gate and negative feedback loops are constructed with the six other sigma factors, two bacteriophage RNA polymerases, and a set of repressors. The circuit dynamics reveal the importance of the global mRNA turnover rate and of passive competition-induced transcriptional regulation. Cell-free reactions can be carried out over long periods of ...

Transcription factor Foxp3 and its protein partners form a complex regulatory network

Abstract: The transcription factor Foxp3 is indispensible for the differentiation and function of regulatory T cells (T(reg) cells). To gain insights into the molecular mechanisms of Foxp3-mediated gene expression, we purified Foxp3 complexes and explored their composition. Biochemical and mass-spectrometric analyses revealed that Foxp3 forms multiprotein complexes of 400-800 kDa or larger and identified 361 associated proteins, ∼30% of which were transcription related. Foxp3 directly regulated expression of a large proportion of the genes encoding its cofactors. Some transcription factor partners of Foxp3 facilitated its expression. Functional analysis of the cooperation of Foxp3 with one such partner, GATA-3, provided additional evidence for a network of transcriptional regulation afforded by Foxp3 and its associates to control distinct aspects of T(reg) cell biology.

Regulation of Gene Expression in Escherichia Coli

Abstract: 1. Introduction: From Physiology to DNA and Back.- 2. RNA Chain Initiation and Promoter Escape by RNA Polymerase.- Promoter Function is Regulated at Two Distinct Phases of Transcription: Promoter Binding and RNA Chain Initiation.- The Biochemistry of the RNA Chain Initiation Phase of Transcription.- Parameters That Describe the RNA Chain Initiation Reaction at Different Promoters.- Factors That Affect the Initiation Reaction: Intrinsic Factors.- Factors That Affect the Initiation Reaction: Extrinsic Factors.- Models for the Mechanism of RNA Chain Initiation: Some Simple Models Do Not Account for What Is Known.- Models for the Mechanism of RNA Chain Initiation-Models Based on Recent Models for RNA Chain Elongation.- 3. Transcription Termination and Its Control.- Termination.- Antitermination.- 4. Codon Context, Translational Step-Times and Attenuation.- Overview.- Attenuation.- Codon Context and Translational Efficiency.- Effects of Codon Pair Bias on Translational Step-Times.- Discussion.- 5. Control by Antisense RNA.- Antisense RNAs Control Diverse Biological Functions.- Antisense RNAs Control Gene Expression at Many Different Post-Transcriptional Levels.- Antisense RNAs Pair to Their Target RNAs by Defined Mechanisms.- Overview.- 6. Translational Control of Gene Expression in E. Coli and Bacteriophage.- Translation Initiation.- Translational Operators.- Translational Repressors.- Mechanisms of Control.- Translational Control and mRNA Processing and/or Degradation.- The Role of Translational Control in Growth Rate Regulation.- Conclusions and Perspectives.- 7. Effects of DNA Supercoiling on Gene Expression.- Synopsis.- The Dependence of Transcription on the Cellular Level of DNA Gyrase and DNA Topoisomerase I.- Mechanistic Considerations.- Supercoiling of the DNA Template by Transcription.- Concluding Remarks.- 8. The HU and IHF Proteins: Accessory Factors for Complex Protein-DNA Assemblies.- Perspective.- Structure.- Interaction with Nucleic Acids.- Control of Intracellular Concentration and Activity.- Participation of IHF and HU in Well-Characterized Biochemical Processes.- Unfinished Business.- 9. The lac and gal Operons Today.- The lac and gal Operons Encode Enzymes of a Continuous Biochemical Pathway.- The Regulatory Circuits and Their Components.- Modulation of Promoters by cAMP*CRP.- Control of P2 by UTP in gal.- Natural Polarity.- Negative Control by Repressor-Operator Interactions.- Epilogue.- 10. The Maltose System.- and Scope.- The Positive Transcriptional Activator MalT.- The Maltose/Maltodextrin Transport System.- The Enzymes of the Maltose System.- Nonclassical Regulatory Phenomena.- Perspectives.- 11. The Phosphoenolpyruvate-Dependent Carbohydrate: Phosphotransferase System (PTS) and Control of Carbon Source Utilization.- Regulatory Phenomena Related to Carbon Source Utilization.- Bacterial Transport Systems and Global Regulatory Networks Form a Unit.- The Bacterial PTS Is a Transport and Signal Transduction System.- IIAGlc of the PTS Is Central to Carbon Catabolite Repression.- IIAGlc, the Regulation of Adenylate Cyclase Activity and of Intracellular cAMP Levels.- Not Only cAMP Levels, but Also CRP Levels Are Essential in Catabolite Repression.- IIAGlc and Inducer Exclusion.- Catabolite Repression and Inducer Exclusion Act in Concert.- Carbon Catabolite Repression through PTS-Control Is Part of a Stimulon.- Concluding Remarks.- 12. The Cap Modulon.- The Long History of CAP.- Cyclic AMP and Gene Expression.- CAP as a Global Regulator: The CAP Modulon.- CAP Binding at Target Promoters and Structural Studies.- Activation by CAP at "Simple"CRP.- Control of P2 by UTP in gal.- Natural Polarity.- Negative Control by Repressor-Operator Interactions.- Epilogue.- 10. The Maltose System.- and Scope.- The Positive Transcriptional Activator MalT.- The Maltose/Maltodextrin Transport System.- The Enzymes of the Maltose System.- Nonclassical Regulatory Phenomena.- Perspectives.- 11. The Phosphoenolpyruvate-Dependent Carbohydrate: Phosphotransferase System (PTS) and Control of Carbon Source Utilization.- Regulatory Phenomena Related to Carbon Source Utilization.- Bacterial Transport Systems and Global Regulatory Networks Form a Unit.- The Bacterial PTS Is a Transport and Signal Transduction System.- IIAGlc of the PTS Is Central to Carbon Catabolite Repression.- IIAGlc, the Regulation of Adenylate Cyclase Activity and of Intracellular cAMP Levels.- Not Only cAMP Levels, but Also CRP Levels Are Essential in Catabolite Repression.- IIAGlc and Inducer Exclusion.- Catabolite Repression and Inducer Exclusion Act in Concert.- Carbon Catabolite Repression through PTS-Control Is Part of a Stimulon.- Concluding Remarks.- 12. The Cap Modulon.- The Long History of CAP.- Cyclic AMP and Gene Expression.- CAP as a Global Regulator: The CAP Modulon.- CAP Binding at Target Promoters and Structural Studies.- Activation by CAP at "Simple" Promoters.- Activation by CAP at Complex Promoters.- CAP as a Repressor and a Co-Repressor.- CAP: Paradigm or Artifact?.- 13. Regulation of Nitrogen Assimilation.- The glnALG(glnA ntrBC) Operon.- The ?54-Dependent Promoter.- Transcriptional Enhancers.- Phosphorylation of NRI.- NRI/NRII as Two-Component Paradigm.- Activation of Transcription.- Response to Nitrogen Availability.- 14. History of the Pho System.- 15. Are the Multiple Signal Transduction Pathways of the Pho Regulon Due to Cross Talk or Cross Regulation?.- Genes for Pi Control of the Pho Regulon.- Transmembrane Signaling by Environmental Pi.- Genes for Pi Independent Controls of the Pho Regulon.- Activation by CreC and Acetyl Phosphate.- Cross Talk, Cross Regulation and a Hypothesis.- Is There Evidence for Cross Regulation?.- Overview and Prospects for Future Studies.- 16. The FNR Modulon and FNR-Regulated Gene Expression.- The Metabolic Arena.- The FNR Modulon.- The FNR Protein and Relationships with CAP.- The DNA-Binding Specificity of FNR.- In Vitro Transcription Activation and Repression.- Transcriptional Organization of Representative Promoters.- Potential FNR Contacts with RNA Polymerase and DNA-Bending.- The Mystery of Redox-Sensing.- Structural and Functional Homologs of FNR.- Concluding Remarks.- 17. The NAR Modulon Systems: Nitrate and Nitrite Regulation of Anaerobic Gene Expression.- Anaerobic Respiration.- The Characterization of the Nar Regulatory System.- Dual Two-Component Regulatory Systems.- The Role of Nitrite in the Nar Regulatory System.- The Sensor Proteins.- The Response Regulators.- Indirect Nitrate Regulation of Gene Expression.- Concluding Remarks.- 18. Regulation of Aerobic and Anaerobic Metabolism by the Arc System.- Identification of the arc Genes.- In Vivo Studies of arc Mutants.- In Vitro Phosphorylation Studies.- The Arc Modulon.- ArcA DNA Binding.- The Arc Stimulus.- Future Studies.- 19. The Porin Regulon: A Paradigm for the Two-Component Regulatory Systems.- Background.- The History of Porin Regulation.- The Structure of the ompB Locus.- The Structure of the ompF and ompC Genes.- The Roles of OmpR and EnvZ.- Phosphorylation and Signal Transduction.- Summary and Conclusions.- 20. The Leucine\Lrp Regulon.- The Leucine-Responsive Regulatory Protein.- Regulation of Lrp Synthesis.- Target Operons of Lrp and Mutant Phenotypes.- Lrp as a Chromosome Organizer.- Molecular Aspects of Lrp Interactions at Individual Promoters.- 21. Adaptive responses to Oxidative Stress: The soxRS and oxyR Regulons.- Reactive Oxygen Species.- Antioxidant Defenses.- Oxidative Stress.- Global Responses to Oxidative Stress.- The soxRS Regulon.- The oxyR Regulon.- Control of Antibiotic Resistance Genes.- 22. The SOS Regulatory System.- and Current Regulatory Model.- Development of the SOS Model.- Recent Developments.- Behavior of the SOS Gene Regulatory Circuitry.- Future Prospects.- 23. Heat Shock Regulation.- Properties of Important Heat Shock Proteins.- Regulation of the ?32-Promoted Heat Shock Response.- A Second Heat Shock Regulon.- The ?54-Promoted Stress Response.- Heat Shock or Stress Responses in Other Eubacteria.- 24. Roles for Energy-Dependent Proteases in Regulatory Cascades.- The Proteases and Their Targets.- Summary and General Conclusions.- 25. Control of rRNA and Ribosome Synthesis.- rRNA Gene Organization.- High Activity of rRNA Synthesis Rates.- Stringent Control.- Growth Rate Dependent Control.- Additional Considerations.- Conclusion and Future Prospects.- 26. Cell Division.- The Cell Division Process.- Essential Cell Division Genes.- Transcriptional Regulation of Cell Division Genes.- Translational Control.- Division Inhibitors.- Cell Division Inhibition by Mutations in Genes That Do Not Code for Cell Division Proteins.- Past, Present and Future.- 27. Regulation of Gene Expression in Stationary Phase.- The ?s Regulon.- RpoS Regulation.

Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains

Abstract: Transcription activator-like effectors are sequence-specific DNA-binding proteins that harbour modular, repetitive DNA-binding domains. Transcription activator-like effectors have enabled the creation of customizable designer transcriptional factors and sequence-specific nucleases for genome engineering. Here we report two improvements of the transcription activator-like effector toolbox for achieving efficient activation and repression of endogenous gene expression in mammalian cells. We show that the naturally occurring repeat-variable diresidue Asn-His (NH) has high biological activity and specificity for guanine, a highly prevalent base in mammalian genomes. We also report an effective transcription activator-like effector transcriptional repressor architecture for targeted inhibition of transcription in mammalian cells. These findings will improve the precision and effectiveness of genome engineering that can be achieved using transcription activator-like effectors. The peptide sequence of transcription activator-like effectors (TALEs) can be customized to tailor the binding of TALEs to specific DNA sequences. Conget al. improve TALE specificity for guanine binding and use a genetic construct based on TALEs to efficiently repress expression of a target gene.

Noncoding Transcription at Enhancers: General Principles and Functional Models

Abstract: Mammalian genomes are extensively transcribed outside the borders of protein-coding genes. Genome-wide studies recently demonstrated that cis-regulatory genomic elements implicated in transcriptional control, such as enhancers and locus-control regions, represent major sites of extragenic noncoding transcription. Enhancer-templated transcripts provide a quantitatively small contribution to the total amount of cellular nonribosomal RNA; nevertheless, the possibility that enhancer transcription and the resulting enhancer RNAs may, in some cases, have functional roles, rather than represent mere transcriptional noise at accessible genomic regions, is supported by an increasing amount of experimental data. In this article we review the current knowledge on enhancer transcription and its functional implications.

Structural basis of transcription initiation.

Abstract: Class II transcription activators function by binding to a DNA site overlapping a core promoter and stimulating isomerization of an initial RNA polymerase (RNAP)–promoter closed complex into a catalytically competent RNAP-promoter open complex. Here, we report a 4.4 angstrom crystal structure of an intact bacterial class II transcription activation complex. The structure comprises Thermus thermophilus transcription activator protein TTHB099 (TAP) [homolog of Escherichia coli catabolite activator protein (CAP)], T. thermophilus RNAP σ A holoenzyme, a class II TAP-dependent promoter, and a ribotetranucleotide primer. The structure reveals the interactions between RNAP holoenzyme and DNA responsible for transcription initiation and reveals the interactions between TAP and RNAP holoenzyme responsible for transcription activation. The structure indicates that TAP stimulates isomerization through simple, adhesive, stabilizing protein-protein interactions with RNAP holoenzyme.

The super elongation complex (SEC) family in transcriptional control

Abstract: The super elongation complex (SEC) consists of the RNA polymerase II (Pol II) elongation factors eleven-nineteen Lys-rich leukaemia (ELL) proteins, positive transcription elongation factor b (P-TEFb) and several frequent mixed lineage leukaemia (MLL) translocation partners. It is one of the most active P-TEFb-containing complexes required for rapid transcriptional induction in the presence or absence of paused Pol II. The SEC was found to regulate the transcriptional elongation checkpoint control (TECC) stage of transcription, and misregulation of this stage is associated with cancer pathogenesis. Recent studies have shown that the SEC belongs to a larger family of SEC-like complexes, which includes SEC-L2 and SEC-L3, each with distinct gene target specificities.

HIV DNA Integration

Abstract: Retroviruses are distinguished from other viruses by two characteristic steps in the viral replication cycle. The first is reverse transcription, which results in the production of a double-stranded DNA copy of the viral RNA genome, and the second is integration, which results in covalent attachment of the DNA copy to host cell DNA. The initial catalytic steps of the integration reaction are performed by the virus-encoded integrase (IN) protein. The chemistry of the IN-mediated DNA breaking and joining steps is well worked out, and structures of IN-DNA complexes have now clarified how the overall complex assembles. Methods developed during these studies were adapted for identification of IN inhibitors, which received FDA approval for use in patients in 2007. At the chromosomal level, HIV integration is strongly favored in active transcription units, which may promote efficient viral gene expression after integration. HIV IN binds to the cellular factor LEDGF/p75, which promotes efficient infection and tethers IN to favored target sites. The HIV integration machinery must also interact with many additional host factors during infection, including nuclear trafficking and pore proteins during nuclear entry, histones during initial target capture, and DNA repair proteins during completion of the DNA joining steps. Models for some of the molecular mechanisms involved have been proposed, but important details remain to be clarified.

SAMHD1 restricts HIV-1 reverse transcription in quiescent CD4(+) T-cells.

Abstract: Background: Quiescent CD4 + T lymphocytes are highly refractory to HIV-1 infection due to a block at reverse transcription. Results: Examination of SAMHD1 expression in peripheral blood lymphocytes shows that SAMHD1 is expressed in both CD4+ and CD8+ T cells at levels comparable to those found in myeloid cells. Treatment of CD4+ T cells with Virus-Like Particles (VLP) containing Vpx results in the loss of SAMHD1 expression that correlates with an increased permissiveness to HIV-1 infection and accumulation of reverse transcribed viral DNA without promoting transcription from the viral LTR. Importantly, CD4 + T-cells from patients with Aicardi-Goutieres Syndrome harboring mutation in the SAMHD1 gene display an increased susceptibility to HIV-1 infection that is not further enhanced by VLP-Vpx-treatment. Conclusion: Here, we identified SAMHD1 as the restriction factor preventing efficient viral DNA synthesis in non-cycling resting CD4 + T-cells. These results highlight the crucial role of SAMHD1 in mediating restriction of HIV-1 infection in quiescent CD4 + T-cells and could impact our understanding of HIV-1 mediated CD4 + T-cell depletion