High resolution mapping of nucleic acid containing complexes in vitro and in situ

Molecular Mechanisms ◽

Heavy Atom ◽

Regulation Of Transcription ◽

High Exposure ◽

Resolution Mapping ◽

Tata Sequence

Structural analysis of combinations of nucleosomes and transcription factors on promoter and enhancer elements is necessary in order to understand the molecular mechanisms responsible for the regulation of transcription initiation. Such complexes are often not amenable to study by high resolution crystallographic techniques. We have been applying electron spectroscopic imaging (ESI) to specific problems in molecular biology related to transcription regulation. There are several advantages that this technique offers in studies of nucleoprotein complexes. First, an intermediate level of spatial resolution can be achieved because heavy atom contrast agents are not necessary. Second, mass and stoichiometric relationships of protein and nucleic acid can be estimated by phosphorus detection, an element in much higher proportions in nucleic acid than protein. Third, wrapping or bending of the DNA by the protein constituents can be observed by phosphorus mapping of the complexes. Even when ESI is used with high exposure of electrons to the specimen, important macromolecular information may be provided. For example, an image of the TATA binding protein (TBP) bound to DNA is shown in the Figure (top panel). It can be seen that the protein distorts the DNA away from itself and much of its mass sits off the DNA helix axis. Moreover, phosphorus and mass estimates demonstrate whether one or two TBP molecules interact with this particular promoter TATA sequence.

Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription

10.1128/mcb.10.6.2832-2839.1990 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2832-2839

Author(s):

A S Ponticelli ◽

K Struhl

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcriptional Activation ◽

Molecular Mechanisms ◽

Initiation Site ◽

Activator Proteins ◽

Nuclear Extracts ◽

Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

High-resolution mapping of cis-regulatory variation in budding yeast

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1717421114 ◽

2017 ◽

Vol 114 (50) ◽

pp. E10736-E10744 ◽

Cited By ~ 22

Author(s):

Ryosuke Kita ◽

Sandeep Venkataram ◽

Yiqi Zhou ◽

Hunter B. Fraser

Keyword(s):

High Resolution ◽

Molecular Mechanisms ◽

Rna Binding ◽

Rna Binding Proteins ◽

Sign Test ◽

Eqtl Mapping ◽

Regulatory Variation ◽

Conserved Genes ◽

Causal Variants ◽

Resolution Mapping

Genetic variants affecting gene-expression levels are a major source of phenotypic variation. The approximate locations of these variants can be mapped as expression quantitative trait loci (eQTLs); however, a major limitation of eQTLs is their low resolution, which precludes investigation of the causal variants and their molecular mechanisms. Here we report RNA-seq and full genome sequences for 85 diverse isolates of the yeast Saccharomyces cerevisiae—including wild, domesticated, and human clinical strains—which allowed us to perform eQTL mapping with 50-fold higher resolution than previously possible. In addition to variants in promoters, we uncovered an important role for variants in 3′UTRs, especially those affecting binding of the PUF family of RNA-binding proteins. The eQTLs are predominantly under negative selection, particularly those affecting essential genes and conserved genes. However, applying the sign test for lineage-specific selection revealed the polygenic up-regulation of dozens of biofilm suppressor genes in strains isolated from human patients, consistent with the key role of biofilms in fungal pathogenicity. In addition, a single variant in the promoter of a biofilm suppressor, NIT3, showed the strongest genome-wide association with clinical origin. Altogether, our results demonstrate the power of high-resolution eQTL mapping in understanding the molecular mechanisms of regulatory variation, as well as the natural selection acting on this variation that drives adaptation to environments, ranging from laboratories to vineyards to the human body.

Integrative In Silico and In Vitro Transcriptomics Analysis Revealed Gene Expression Changes and Oncogenic Features of Normal Cholangiocytes after Chronic Alcohol Exposure

International Journal of Molecular Sciences ◽

10.3390/ijms20235987 ◽

2019 ◽

Vol 20 (23) ◽

pp. 5987

Author(s):

Suthipong Chujan ◽

Tawit Suriyo ◽

Jutamaad Satayavivad

Keyword(s):

Gene Expression ◽

Rna Polymerase Ii ◽

Molecular Mechanisms ◽

Function Analysis ◽

Alcohol Exposure ◽

Gene Expression Omnibus ◽

Regulation Of Transcription ◽

Chronic Alcohol ◽

Chronic Alcohol Exposure

Cholangiocarcinoma (CCA) is a malignant tumor originating from cholangiocyte. Prolonged alcohol consumption has been suggested as a possible risk factor for CCA, but there is no information about alcohol’s mechanisms in cholangiocyte. This study was designed to investigate global transcriptional alterations through RNA-sequencing by using chronic alcohol exposure (20 mM for 2 months) in normal human cholangiocyte MMNK-1 cells. To observe the association of alcohol induced CCA pathogenesis, we combined differentially expressed genes (DEGs) with computational bioinformatics of CCA by using publicly gene expression omnibus (GEO) datasets. For biological function analysis, Gene ontology (GO) analysis showed biological process and molecular function related to regulation of transcription from RNA polymerase II promoter, while cellular component linked to the nucleoplasm. KEGG pathway presented pathways in cancer that were significantly enriched. From KEGG result, we further examined the oncogenic features resulting in chronic alcohol exposure, enhanced proliferation, and migration through CCND-1 and MMP-2 up-regulation, respectively. Finally, combined DEGs were validated in clinical data including TCGA and immunohistochemistry from HPA database, demonstrating that FOS up-regulation was related to CCA pathogenesis. This study is the first providing more information and molecular mechanisms about global transcriptome alterations and oncogenic enhancement of chronic alcohol exposure in normal cholangiocytes.

Molecular Mechanisms Underlying the Positive Stringent Response of the Bacillus subtilis ilv-leu Operon, Involved in the Biosynthesis of Branched-Chain Amino Acids

Journal of Bacteriology ◽

10.1128/jb.00606-08 ◽

2008 ◽

Vol 190 (18) ◽

pp. 6134-6147 ◽

Cited By ~ 33

Author(s):

Shigeo Tojo ◽

Takenori Satomura ◽

Kanako Kumamoto ◽

Kazutake Hirooka ◽

Yasutaro Fujita

Keyword(s):

Amino Acids ◽

Bacillus Subtilis ◽

Molecular Mechanisms ◽

Stringent Response ◽

Branched Chain Amino Acids ◽

Base Substitution ◽

Branched Chain

ABSTRACT Branched-chain amino acids are the most abundant amino acids in proteins. The Bacillus subtilis ilv-leu operon is involved in the biosynthesis of branched-chain amino acids. This operon exhibits a RelA-dependent positive stringent response to amino acid starvation. We investigated this positive stringent response upon lysine starvation as well as decoyinine treatment. Deletion analysis involving various lacZ fusions revealed two molecular mechanisms underlying the positive stringent response of ilv-leu, i.e., CodY-dependent and -independent mechanisms. The former is most likely triggered by the decrease in the in vivo concentration of GTP upon lysine starvation, GTP being a corepressor of the CodY protein. So, the GTP decrease derepressed ilv-leu expression through detachment of the CodY protein from its cis elements upstream of the ilv-leu promoter. By means of base substitution and in vitro transcription analyses, the latter (CodY-independent) mechanism was found to comprise the modulation of the transcription initiation frequency, which likely depends on fluctuation of the in vivo RNA polymerase substrate concentrations after stringent treatment, and to involve at least the base species of adenine at the 5′ end of the ilv-leu transcript. As discussed, this mechanism is presumably distinct from that for B. subtilis rrn operons, which involves changes in the in vivo concentration of the initiating GTP.

Challenges and perspectives for structural biology of lncRNAs—the example of the Xist lncRNA A-repeats

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjz086 ◽

2019 ◽

Vol 11 (10) ◽

pp. 845-859 ◽

Cited By ~ 10

Author(s):

Alisha N Jones ◽

Michael Sattler

Keyword(s):

High Resolution ◽

Structural Biology ◽

Recent Progress ◽

Molecular Mechanisms ◽

Rna Binding ◽

Rna Binding Proteins ◽

Structural Features ◽

Molecular Features

Abstract Following the discovery of numerous long non-coding RNA (lncRNA) transcripts in the human genome, their important roles in biology and human disease are emerging. Recent progress in experimental methods has enabled the identification of structural features of lncRNAs. However, determining high-resolution structures is challenging as lncRNAs are expected to be dynamic and adopt multiple conformations, which may be modulated by interaction with protein binding partners. The X-inactive specific transcript (Xist) is necessary for X inactivation during dosage compensation in female placental mammals and one of the best-studied lncRNAs. Recent progress has provided new insights into the domain organization, molecular features, and RNA binding proteins that interact with distinct regions of Xist. The A-repeats located at the 5′ end of the transcript are of particular interest as they are essential for mediating silencing of the inactive X chromosome. Here, we discuss recent progress with elucidating structural features of the Xist lncRNA, focusing on the A-repeats. We discuss the experimental and computational approaches employed that have led to distinct structural models, likely reflecting the intrinsic dynamics of this RNA. The presence of multiple dynamic conformations may also play an important role in the formation of the associated RNPs, thus influencing the molecular mechanism underlying the biological function of the Xist A-repeats. We propose that integrative approaches that combine biochemical experiments and high-resolution structural biology in vitro with chemical probing and functional studies in vivo are required to unravel the molecular mechanisms of lncRNAs.

New Approaches for Mapping Epigenome Dynamics

Blood ◽

10.1182/blood.v126.23.sci-21.sci-21 ◽

2015 ◽

Vol 126 (23) ◽

pp. SCI-21-SCI-21

Author(s):

Steven Henikoff

Keyword(s):

Transcription Factors ◽

Rna Polymerase Ii ◽

Conflicts Of Interest ◽

Regulation Of Transcription ◽

Base Pairs ◽

Simple Method ◽

Genome Wide ◽

Resolution Mapping

Abstract The protein complexes that package our genomes must be mobilized for active processes to occur, including replication and transcription, but until recently we have only had a static, low resolution view of the "epigenome". Genomes are packaged into nucleosomes, octamers of four core histones wrapped by 147 base pairs of DNA. Nucleosomes present obstacles to transcription, which over genes is the RNA Polymerase II (RNAPII) complex, and one current challenge is to understand what happens to a nucleosome when RNAPII transcribes through the DNA that it occupies. We study this process by developing methods for following nucleosomes as they are evicted and replaced. Among the factors that we have implicated in the process is torsional stress, which we can now measure genome-wide. RNAPII movement can unwrap nucleosomes and thus destabilize them, causing them to be occasionally evicted and replaced. Interestingly, we find that destabilization of nucleosomes during transcription is enhanced by anthracycline compounds, widely used chemotherapeutic drugs that intercalate between DNA base pairs, thus suggesting a new mechanism for cell killing during chemotherapy. We are also interested in what happens to RNAPII during its encounter with a nucleosomes. In vitro, RNAPII cannot transcribe completely through a nucleosome, but rather stalls as it tries to unwrap the DNA from around the core. We have been studying this process in vivo, and have developed a simple method for precisely mapping RNAPII genome-wide. We have used this method to show exactly where RNAPII stalls as it unwraps a nucleosome in vivo, surprisingly in a different place in vivo from where it stalls in vitro. We also have discovered that a variant histone, H2A.Z, which is found in essentially all eukaryotes, helps to reduce the nucleosome barrier to transcription, and in this way may modulate transcription. Other protein components of the epigenome involved in dynamic processes are nucleosome remodelers, which use the energy of ATP to slide or even evict nucleosomes from DNA. Some remodelers help RNAPII get started and others help it overcome the nucleosome barrier to transcription, and by mapping them at base-pair resolution, we can gain insight into how they act. We have also applied our high-resolution mapping tools to transcription factors, which bind DNA at specific sites to regulate transcription and other processes. Our ability to achieve high spatial and temporal resolution mapping of the binding and action of nucleosomes, transcription factors, remodelers and RNAPII provides us with a detailed picture of epigenome dynamics. By using these tools we are beginning to understand how DNA sequence and conformation are recognized for regulation of transcription and other epigenomic processes. Disclosures No relevant conflicts of interest to declare.

A nuclease-hypersensitive element of the human c-myc promoter interacts with a transcription initiation factor

10.1128/mcb.9.11.5123-5133.1989 ◽

1989 ◽

Vol 9 (11) ◽

pp. 5123-5133

Author(s):

E H Postel ◽

S E Mango ◽

S J Flint

Keyword(s):

Transcription Factor ◽

Molecular Mechanisms ◽

Initiation Factor ◽

Mobility Shift ◽

Cis Acting ◽

Factor Binding ◽

Myc Oncogene ◽

Transcription In Vitro

Transcription of the human c-myc oncogene is elaborately regulated, but the relevant molecular mechanisms are not yet understood. To begin to define elements and enzyme systems responsible for c-myc transcription in vitro, we partially purified a transcription factor essential for efficient and accurate in vitro initiation from the principal myc promoter, P2. DNA mobility shift assays located the factor binding domain at -142 to -115 with respect to the P1 promoter. This region contains pur/pyr sequences (predominantly purines in one strand), nuclease-hypersensitive sites (U. Siebenlist, L. Henninghausen, J. Battey, and P. Leder, Cell 37:381-391, 1984; C. Boles and M. Hogan, Biochemistry 26:367-376, 1987), and a triple-helix-forming element (M. Cooney, G. Czernuszewicz, E. Postel, S. Flint, and M. Hogan, Science 241:456-459, 1988). Methylation interference mapping established that the factor, termed PuF, directly contacts the repeated palindromic sequence GGGTGGG of the -142/-115 element. The interaction of PuF with this cis-acting element is necessary for P2 transcription in vitro, for (i) deletion of this 5' region from the myc promoter greatly reduced transcription efficiency and (ii) a synthetic duplex oligonucleotide corresponding to the -142/-115 sequence completely repressed c-myc transcription in the presence of the partially purified factor. These observations lend support to the hypothesis that pur/pyr sequences perform important biological roles in the regulation of c-myc gene expression, most likely by serving as transcription factor binding sites.

In vitro transcription of a Drosophila U1 small nuclear RNA gene requires TATA box-binding protein and two proximal cis-acting elements with stringent spacing requirements

10.1128/mcb.13.9.5918-5927.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5918-5927

Author(s):

Z Zamrod ◽

C M Tyree ◽

Y Song ◽

W E Stumph

Keyword(s):

Rna Polymerase Ii ◽

Tata Box ◽

Start Site ◽

Small Nuclear Rna ◽

Wild Type ◽

Nuclear Rna ◽

Tata Sequence ◽

Tata Box Binding Protein

Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, is located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduced transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increased the efficiency of the U1 promoter and simultaneously relieved the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter had no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter.

The Yeast FACT Complex Has a Role in Transcriptional Initiation

10.1128/mcb.25.14.5812-5822.2005 ◽

2005 ◽

Vol 25 (14) ◽

pp. 5812-5822 ◽

Cited By ~ 58

Author(s):

Debabrata Biswas ◽

Yaxin Yu ◽

Matthew Prall ◽

Tim Formosa ◽

David J. Stillman

Keyword(s):

Complex Formation ◽

Dependent Manner ◽

Saga Complex ◽

Transcriptional Initiation ◽

A Subunit ◽

Tata Sequence ◽

Synthetically Lethal

ABSTRACT A crucial step in eukaryotic transcriptional initiation is recognition of the promoter TATA by the TATA-binding protein (TBP), which then allows TFIIA and TFIIB to be recruited. However, nucleosomes block the interaction between TBP and DNA. We show that the yeast FACT complex (yFACT) promotes TBP binding to a TATA box in chromatin both in vivo and in vitro. The SPT16 gene encodes a subunit of yFACT, and we show that certain spt16 mutations are synthetically lethal with TBP mutants. Some of these genetic defects can be suppressed by TFIIA overexpression, strongly suggesting a role for yFACT in TBP-TFIIA complex formation in vivo. Mutations in the TOA2 subunit of TFIIA that disrupt TBP-TFIIA complex formation in vitro are also synthetically lethal with spt16. In some cases this spt16 toa2 lethality is suppressed by overexpression of TBP or the Nhp6 architectural transcription factor that is also a component of yFACT. The Spt3 protein in the SAGA complex has been shown to regulate TBP binding at certain promoters, and we show that some spt16 phenotypes can be suppressed by spt3 mutations. Chromatin immunoprecipitations show TBP binding to promoters is reduced in single spt16 and spt3 mutants but increases in the spt16 spt3 double mutant, reflecting the mutual suppression seen in the genetic assays. Finally, in vitro studies show that yFACT promotes TBP binding to a TATA sequence within a reconstituted nucleosome in a TFIIA-dependent manner. Thus, yFACT functions in establishing transcription initiation complexes in addition to the previously described role in elongation.