scholarly journals Oscillating and stable genome topologies underlie hepatic physiological rhythms during the circadian cycle

PLoS Genetics ◽  
2021 ◽  
Vol 17 (2) ◽  
pp. e1009350
Author(s):  
Jérôme Mermet ◽  
Jake Yeung ◽  
Felix Naef
Keyword(s):  
Rna Polymerase Ii ◽  
Clock Genes ◽  
Gene Promoter ◽  
Chromatin Interaction ◽  
Gene Promoters ◽  
Chromatin Modifications ◽  
Chromosome Conformation ◽  
Time Resolved ◽  
Liver Physiology ◽  
Stable Genome

The circadian clock drives extensive temporal gene expression programs controlling daily changes in behavior and physiology. In mouse liver, transcription factors dynamics, chromatin modifications, and RNA Polymerase II (PolII) activity oscillate throughout the 24-hour (24h) day, regulating the rhythmic synthesis of thousands of transcripts. Also, 24h rhythms in gene promoter-enhancer chromatin looping accompany rhythmic mRNA synthesis. However, how chromatin organization impinges on temporal transcription and liver physiology remains unclear. Here, we applied time-resolved chromosome conformation capture (4C-seq) in livers of WT and arrhythmic Bmal1 knockout mice. In WT, we observed 24h oscillations in promoter-enhancer loops at multiple loci including the core-clock genes Period1, Period2 and Bmal1. In addition, we detected rhythmic PolII activity, chromatin modifications and transcription involving stable chromatin loops at clock-output gene promoters representing key liver function such as glucose metabolism and detoxification. Intriguingly, these contacts persisted in clock-impaired mice in which both PolII activity and chromatin marks no longer oscillated. Finally, we observed chromatin interaction hubs connecting neighbouring genes showing coherent transcription regulation across genotypes. Thus, both clock-controlled and clock-independent chromatin topology underlie rhythmic regulation of liver physiology.

scholarly journals Oscillating and stable genome topologies underlie hepatic physiological rhythms during the circadian cycle

2020 ◽  
Author(s):  
Jérôme Mermet ◽  
Jake Yeung ◽  
Félix Naef
Keyword(s):  
Rna Polymerase Ii ◽  
Clock Genes ◽  
Gene Promoter ◽  
Chromatin Interaction ◽  
Gene Promoters ◽  
Chromatin Modifications ◽  
Chromosome Conformation ◽  
Time Resolved ◽  
Liver Physiology ◽  
Stable Genome

AbstractThe circadian clock drives extensive temporal gene expression programs controlling daily changes in behavior and physiology. In mouse liver, transcription factors dynamics, chromatin modifications, and RNA Polymerase II (PolII) activity oscillate throughout the 24-hour (24h) day, regulating the rhythmic synthesis of thousands of transcripts. Also, 24h rhythms in gene promoter-enhancer chromatin looping accompany rhythmic mRNA synthesis. However, how chromatin organization impinges on temporal transcription and liver physiology remains unclear. Here, we applied time-resolved chromosome conformation capture (4C-seq) in livers of WT and arrhythmic Bmal1 knockout mice. In WT, we observed 24h oscillations in promoter-enhancer contact at multiple loci including the core-clock genes Period1, Period2 and Bmal1. In addition, we detected rhythmic PolII activity, chromatin modifications and transcription involving stable chromatin loops at gene promoters representing key liver function such as glucose and lipid metabolism and detoxification. Intriguingly, these contacts persisted in clock-impaired mice in which both PolII activity and chromatin marks no longer oscillated. Finally, we observed chromatin interaction hubs connecting neighbouring genes showing coherent transcription regulation across genotypes. Thus, both clock-controlled and clock-independent chromatin topology underlie rhythmic regulation of liver physiology.

scholarly journals Enzymatic Protein Biopolymers as a Tool to Synthetize Eukaryotic Messenger Ribonucleic Acid (mRNA) with Uses in Vaccination, Immunotherapy and Nanotechnology

Polymers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 1633
Author(s):  
Fabiola Urbina ◽  
Sebastián Morales-Pison ◽  
Edio Maldonado
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Gene Promoter ◽  
Test Tube ◽  
Gene Promoters ◽  
Human Beings ◽  
Promoter Elements ◽  
Messenger Ribonucleic Acid

Multi-subunit enzymes are protein biopolymers that are involved in many cellular processes. The enzyme that carries out the process of transcription of mRNAs is RNA polymerase II (RNAPII), which is a multi-subunit enzyme in eukaryotes. This protein biopolymer starts the transcription from specific sites and is positioned by transcription factors, which form a preinitiation complex (PIC) on gene promoters. To recognize and position the RNAPII and the transcription factors on the gene promoters are needed specific DNA sequences in the gene promoters, which are named promoter elements. Those gene promoter elements can vary and therefore several kinds of promoters exist, however, it appears that all promoters can use a similar pathway for PIC formation. Those pathways are discussed in this review. The in vitro transcribed mRNA can be used as vaccines to fight infectious diseases, e.g., in immunotherapy against cancer and in nanotechnology to deliver mRNA for a missing protein into the cell. We have outlined a procedure to produce an mRNA vaccine against the SARS-CoV-2 virus, which is the causing agent of the big pandemic, COVID-19, affecting human beings all over the world. The potential advantages of using eukaryotic RNAPII to synthetize large transcripts are outlined and discussed. In addition, we suggest a method to cap the mRNA at the 5′ terminus by using enzymes, which might be more effective than cap analogs. Finally, we suggest the construction of a future multi-talented RNAPII, which would be able to synthetize large mRNA and cap them in the test tube.

scholarly journals Activity of chimeric U small nuclear RNA (snRNA)/mRNA genes in transfected protoplasts of Nicotiana plumbaginifolia: U snRNA 3'-end formation and transcription initiation can occur independently in plants.

1993 ◽  
Vol 13 (10) ◽  
pp. 6403-6415 ◽  
Author(s):  
S Connelly ◽  
W Filipowicz
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Gene Promoter ◽  
Nicotiana Plumbaginifolia ◽  
Gene Promoters ◽  
Coding Region ◽  
Pol Ii ◽  
U2 Snrna ◽  
Snrna Gene

Formation of the 3' ends of RNA polymerase II (Pol II)-specific U small nuclear RNAs (U snRNAs) in vertebrate cells is dependent upon transcription initiation from the U snRNA gene promoter. Moreover, U snRNA promoters are unable to direct the synthesis of functional polyadenylated mRNAs. In this work, we have investigated whether U snRNA 3'-end formation and transcription initiation are also coupled in plants. We have first characterized the requirements for 3'-end formation of an Arabidopsis U2 snRNA expressed in transfected protoplasts of Nicotiana plumbaginifolia. We found that the 3'-end-adjacent sequence CA (N)3-10AGTNNAA, conserved in plant Pol II-specific U snRNA genes, is essential for the 3'-end formation of U2 transcripts and, similar to the vertebrate 3' box, is highly tolerant to mutation. The 3'-flanking regions of an Arabidopsis U5 and a maize U2 snRNA gene can effectively substitute for the Arabidopsis U2 3'-end formation signal, indicating that these signals are functionally equivalent among different Pol II-transcribed snRNA genes. The plant U snRNA 3'-end formation signal can be recognized irrespective of whether transcription initiation occurs at U snRNA or mRNA gene promoters, although efficiency of 3' box utilization is higher when transcription initiation occurs at the U snRNA promoter. Moreover, transcripts initiated from the U2 gene promoter can be spliced and polyadenylated. Transcription from a Pol III-specific plant U snRNA gene promoter is not compatible with polyadenylation. Finally, we reveal that initiation at a Pol II-specific plant U snRNA gene promoter can occur in the absence of the snRNA coding region and a functional snRNA 3'-end formation signal, demonstrating that these sequences play no role in determining the RNA polymerase specificity of plant U snRNA genes.

scholarly journals Development of a Virus-Based Reporter System for Functional Analysis of Plant rRNA Gene Promoter

2021 ◽  
Vol 12 ◽  
Author(s):  
Li Xu ◽  
Zhiying Li ◽  
Sheng Wang
Keyword(s):  
Rna Polymerase Ii ◽  
Reporter Gene ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Cauliflower Mosaic Virus ◽  
Control Element ◽  
35S Promoter ◽  
Rrna Gene ◽  
Gene Promoters ◽  
Reporter System

Reporter gene-based expression systems have been intensively used in plants for monitoring the activity of gene promoters. However, rRNA transcripts are unable to efficiently express a reporter gene due to a lack of a 5' cap. Because of this obstacle, plant rRNA gene promoters are less well characterized to this day. We developed a virus-based reporter system to characterize the Nicotiana benthamiana rRNA (NbrRNA) gene promoter. The system utilizes the cap-independent translation strategy of viral genomic mRNA and uses the virus-expressed green fluorescent protein (GFP) as an indicator of the rRNA gene promoter activity in virus-infected plants. Based on the reporter system, some characteristics of the N. benthamiana rRNA gene promoter were revealed. The results showed that the strength of the NbrRNA gene promoter was lower than that of the cauliflower mosaic virus (CaMV) 35S promoter, a well-characterized polymerase II promoter. The sequences between −77 and +42 are sufficient for the NbrRNA gene promoter-mediated transcription and the NbrRNA gene promoter may lack the functional upstream control element (UCE). Interestingly, NbrRNA gene promoter activity was increased when the 35S enhancer was introduced. An intron-excision mediated assay revealed that the NbrRNA gene promoter can be inefficiently used by RNA polymerase II in N. benthamiana cells. This virus-based reporter system is easier to operate and more convenient when compared with the previously Pol I promoter assays. And it offers a promising solution to analyzing the functional architecture of plant rRNA gene promoter.

scholarly journals Chromosomal architecture and placental expression of the human growth hormone gene family are targeted by pre-pregnancy maternal obesity

2018 ◽  
Vol 315 (4) ◽  
pp. E435-E445 ◽  
Author(s):  
Yan Jin ◽  
Hana Vakili ◽  
Song Yan Liu ◽  
Savas Menticoglou ◽  
Margaret E. Bock ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Rna Polymerase Ii ◽  
Maternal Obesity ◽  
Prolactin Receptor ◽  
Chromosome 17 ◽  
Growth Hormone Gene ◽  
Gene Promoters ◽  
Obese Women ◽  
Chromosome Conformation ◽  
Chromosomal Architecture

The human (h) placental lactogenic hormone chorionic somatomammotropin (CS) is highly produced during pregnancy and acts as a metabolic adaptor in response to maternal insulin resistance. Maternal obesity can exacerbate this “resistance”, and a >75% decrease in CS RNA levels was observed in term placentas from obese vs. lean women. The genes coding for hCS ( hCS-A and hCS-B) and placental growth hormone ( hGH-V) as well as the hCS-L pseudogene and pituitary growth hormone (GH) gene ( hGH-N) are located at a single locus on chromosome 17. Three remote hypersensitive sites (HS III–V) located >28 kb upstream of hGH-N as well as local hCS gene promoter and enhancer regions are implicated in hCS gene expression. A placenta-specific chromosomal architecture, including interaction between HS III–V and hCS but not hGH gene promoters, was detected in placentas from lean women (BMI <25 kg/m2) by using the chromosome conformation capture assay. This architecture was disrupted by pre-pregnancy maternal obesity (BMI >35 kg/m2), resulting in a predominant interaction between HS III and the hGH-N promoter, which was also observed in nonplacental tissues. This was accompanied by a decrease in hCS levels, which was consistent with reduced RNA polymerase II and CCAAT/enhancer-binding protein-β association with individual hCS promoter and enhancer sequences, respectively. Thus, pre-pregnancy maternal obesity disrupts the placental hGH/CS gene locus chromosomal architecture. However, based on data from obese women who develop GDM, insulin treatment partially recapitulates the chromosomal architecture seen in lean women and positively affects hCS production, presumably facilitating prolactin receptor-related signaling by hCS.

Activity of chimeric U small nuclear RNA (snRNA)/mRNA genes in transfected protoplasts of Nicotiana plumbaginifolia: U snRNA 3'-end formation and transcription initiation can occur independently in plants

1993 ◽  
Vol 13 (10) ◽  
pp. 6403-6415
Author(s):  
S Connelly ◽  
W Filipowicz
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Gene Promoter ◽  
Nicotiana Plumbaginifolia ◽  
Gene Promoters ◽  
Coding Region ◽  
Pol Ii ◽  
U2 Snrna ◽  
Snrna Gene

Formation of the 3' ends of RNA polymerase II (Pol II)-specific U small nuclear RNAs (U snRNAs) in vertebrate cells is dependent upon transcription initiation from the U snRNA gene promoter. Moreover, U snRNA promoters are unable to direct the synthesis of functional polyadenylated mRNAs. In this work, we have investigated whether U snRNA 3'-end formation and transcription initiation are also coupled in plants. We have first characterized the requirements for 3'-end formation of an Arabidopsis U2 snRNA expressed in transfected protoplasts of Nicotiana plumbaginifolia. We found that the 3'-end-adjacent sequence CA (N)3-10AGTNNAA, conserved in plant Pol II-specific U snRNA genes, is essential for the 3'-end formation of U2 transcripts and, similar to the vertebrate 3' box, is highly tolerant to mutation. The 3'-flanking regions of an Arabidopsis U5 and a maize U2 snRNA gene can effectively substitute for the Arabidopsis U2 3'-end formation signal, indicating that these signals are functionally equivalent among different Pol II-transcribed snRNA genes. The plant U snRNA 3'-end formation signal can be recognized irrespective of whether transcription initiation occurs at U snRNA or mRNA gene promoters, although efficiency of 3' box utilization is higher when transcription initiation occurs at the U snRNA promoter. Moreover, transcripts initiated from the U2 gene promoter can be spliced and polyadenylated. Transcription from a Pol III-specific plant U snRNA gene promoter is not compatible with polyadenylation. Finally, we reveal that initiation at a Pol II-specific plant U snRNA gene promoter can occur in the absence of the snRNA coding region and a functional snRNA 3'-end formation signal, demonstrating that these sequences play no role in determining the RNA polymerase specificity of plant U snRNA genes.

scholarly journals Free energy based high-resolution modeling of CTCF-mediated chromatin loops for human genome

2017 ◽  
Author(s):  
Wayne Dawson ◽  
Dariusz Plewczynski
Keyword(s):  
Free Energy ◽  
Rna Polymerase Ii ◽  
Human Genome ◽  
Interaction Analysis ◽  
Minimum Free Energy ◽  
Chromatin Interaction ◽  
Specific Method ◽  
Genomic Distance ◽  
Chromosome Conformation ◽  
Chromatin Loops

AbstractIn recent years, chromatin has been found to have considerable structural organization in the human genome with diverse parts of the chromatin interacting with each other to form what have been termed topologically associated domains (TADs). Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) is a recent protein-specific method that measures these chromatin interactions via specific interactions such as CTCF-cohesin binding proteins or RNA polymerase II interactions. Unlike high-throughput chromosome conformation capture (Hi-C), which measures unspecific binding (all against all), ChIA-PET measures specific protein-protein contact interactions; hence physical bonds that reflect binding free energies. In this work, a thermodynamic method for computing the stability and dynamics of chromatin loops is proposed. The CTCF-mediated interactions, as observed in ChIA-PET experiments for human B-lymphoblastoid cells, are evaluated in terms of a chain folding polymer model and the experimentally observed frequency of contacts within the chromatin regions. To estimate the optimal free energy and a Boltzmann distribution of suboptimal structures, the approach uses dynamic programming with methods to handle degeneracy and heuristics to compute parallel and antiparallel chain stems and pseudoknots. Moreover, multiple loops mediated by CTCF protein binding that connects together more than one chain into multimeric islands are simulated using the model. Based on the thermodynamic properties of those topological three-dimensional structures, we predict the correlation between the relative activity of chromatin loop and the Boltzmann probability, or the minimum free energy, depending also on its genomic length. The results show that segments of chromatin where the structures show a more stable minimum free energy (for a given genomic distance) tend to be inactive, whereas structures that have lower stability in the minimum free energy (with the same genomic distance) tend to be active.

scholarly journals Real-Time Interaction between TVR and the TATA Box of the Human Triosephosphate Isomerase Gene Promoter in the Norm and Pathology

Acta Naturae ◽  
2014 ◽  
Vol 6 (2) ◽  
pp. 36-40 ◽  
Author(s):  
O. V. Arkova ◽  
N. A. Kuznetsov ◽  
O. S. Fedorova ◽  
N. A. Kolchanov ◽  
L. K. Savinkova
Keyword(s):  
Complex Formation ◽  
Rna Polymerase Ii ◽  
Real Time ◽  
Triosephosphate Isomerase ◽  
Gene Promoter ◽  
Hereditary Diseases ◽  
Tata Box ◽  
Gene Promoters ◽  
Wild Type ◽  
Increased Risk

The TATA-binding protein (ТВР) is a key part of the transcription complex of RNA polymerase II. Alone or as a part of the basal transcription factor TFIID, TBP binds the TATA box located in the core region of the TATA-containing promoters of class II genes. Previously, we studied the effects of single nucleotide polymorphisms (SNPs) on ТВР/ТАТА-box interactions using gel retardation assay. It was demonstrated that most SNPs in the ТАТА boxes of some human gene promoters cause a 2- to 4-fold decrease in ТВР/ТАТА affinity, which is associated with an increased risk of hereditary diseases, such as thalassemias of diverse severity, hemophilia B Leyden, myocardial infarction, thrombophlebitis, lung cancer, etc. In this work, the process of ТВР/ТАТА complex formation has been studied in real time by a stopped-flow technique using recombinant human TBP and duplexes, which were identical to the TATA box of the wild-type and a SNP-containing triosephosphate isomerase gene promoter and were fluorescently labeled by the Cy3/Cy5 FRET pair. It has been demonstrated for the first time that real-time binding of ТВР to the TATA box of the TPI gene promoter is complete within 10 s and is described by a single-stage kinetic model. The complex formation of ТВР with the wild-type TATA box occurs 5.5 times faster and the complex dissociation occurs 31 times slower compared with the SNPcontaining TATA box. Within the first seconds of the interaction, ТВР binds to and simultaneously bends the TATA box. Importantly, the TATA box of the wild-type TPI gene promoter requires lower TBP concentrations compared to the TATA box containing the -24Т G SNP, which is associated with neurological and muscular disorders, cardiomyopathy, and other diseases.

scholarly journals m6A RNA methylation regulates promoter proximal pausing of RNA Polymerase II

2020 ◽  
Author(s):  
Junaid Akhtar ◽  
Yoan Renaud ◽  
Steffen Albrecht ◽  
Yad Ghavi-Helm ◽  
Jean-Yves Roignant ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Domain ◽  
Gene Promoter ◽  
Rna Modification ◽  
Gene Promoters ◽  
Rna Methylation ◽  
Control Gene Expression ◽  
M6a Rna Methylation ◽  
Rnap Ii

AbstractRNA Polymerase II (RNAP II) pausing is essential to precisely control gene expression and is critical for development of metazoans. Here, we show that the m6A RNA modification regulates promoter-proximal RNAP II pausing. The m6A methyltransferase complex (MTC), with the nuclear reader Ythdc1, are recruited to gene promoters. Depleting the m6A MTC leads to a decrease in RNAP II pause release and in Ser2P occupancy on the gene body, and affects nascent RNA transcription. Tethering Mettl3 to a heterologous gene promoter is sufficient to increase RNAP II pause release, an effect that relies on its m6A catalytic domain. Collectively, our data reveal an important link between RNAP II pausing and the m6A RNA modification, thus adding another layer to m6A-mediated gene regulation.

scholarly journals 3D reconstruction of genomic regions from sparse interaction data

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Julen Mendieta-Esteban ◽  
Marco Di Stefano ◽  
David Castillo ◽  
Irene Farabella ◽  
Marc A Marti-Renom
Keyword(s):  
3D Models ◽  
Genomic Region ◽  
Gene Promoters ◽  
Chromosome Conformation ◽  
The Matrix ◽  
Chromatin Structural ◽  
A Cell ◽  
Similar Accuracy ◽  
Genomic Regions ◽  
Interaction Matrices

Abstract Chromosome conformation capture (3C) technologies measure the interaction frequency between pairs of chromatin regions within the nucleus in a cell or a population of cells. Some of these 3C technologies retrieve interactions involving non-contiguous sets of loci, resulting in sparse interaction matrices. One of such 3C technologies is Promoter Capture Hi-C (pcHi-C) that is tailored to probe only interactions involving gene promoters. As such, pcHi-C provides sparse interaction matrices that are suitable to characterize short- and long-range enhancer–promoter interactions. Here, we introduce a new method to reconstruct the chromatin structural (3D) organization from sparse 3C-based datasets such as pcHi-C. Our method allows for data normalization, detection of significant interactions and reconstruction of the full 3D organization of the genomic region despite of the data sparseness. Specifically, it builds, with as low as the 2–3% of the data from the matrix, reliable 3D models of similar accuracy of those based on dense interaction matrices. Furthermore, the method is sensitive enough to detect cell-type-specific 3D organizational features such as the formation of different networks of active gene communities.

Sign in / Sign up

Export Citation Format

Share Document