scholarly journals Cell-free transcriptional regulation via nucleic-acid-based transcription factors

2019 ◽  
Author(s):  
Leo Y.T. Chou ◽  
William M. Shih
Keyword(s):  
Transcription Factors ◽  
Nucleic Acid ◽  
Gene Networks ◽  
De Novo ◽  
Dna Nanotechnology ◽  
Regulatory Elements ◽  
Nucleic Acid Hybridization ◽  
Artificial Transcription Factors ◽  
Free Gene

AbstractCells execute complex transcriptional programs by deploying distinct protein regulatory assemblies that interact with cis-regulatory elements throughout the genome. Using concepts from DNA nanotechnology, we synthetically recapitulated this feature in cell-free gene networks actuated by T7 RNA polymerase (RNAP). Our approach involves engineering nucleic-acid hybridization interactions between a T7 RNAP site-specifically functionalized with single-stranded DNA (ssDNA), templates displaying cis-regulatory ssDNA domains, and auxiliary nucleic-acid assemblies acting as artificial transcription factors (TFs). By relying on nucleic-acid hybridization, de novo regulatory assemblies can be computationally designed to emulate features of protein-based TFs, such as cooperativity and combinatorial binding, while offering unique advantages such as programmability, chemical stability, and scalability. We illustrate the use of nucleic-acid TFs to implement transcriptional logic, cascading, feedback, and multiplexing. This framework will enable rapid prototyping of increasingly complex in vitro genetic devices for applications such as portable diagnostics, bio-analysis, and the design of adaptive materials.

scholarly journals HisTrader: A Tool to Identify Nucleosome Free Regions from ChIP-Seq of Histone Post-Translational Modifications

2020 ◽  
Author(s):  
Yifei Yan ◽  
Ansley Gnanapragasam ◽  
Swneke Bailey
Keyword(s):  
Transcription Factors ◽  
Histone Modification ◽  
Dna Sequence ◽  
De Novo ◽  
Cancer Cell Line ◽  
Regulatory Elements ◽  
Lung Cancer Cell Line ◽  
Sequence Length ◽  
Post Translational Modifications ◽  
Disease States

ABSTRACTMotivationChromatin immuno-precipitation sequencing (ChIP-Seq) of histone post-translational modifications coupled with de novo motif elucidation and enrichment analyses can identify transcription factors responsible for orchestrating transitions between cell-and disease-states. However, the identified regulatory elements can span several kilobases (kb) in length, which complicates motif-based analyses. Restricting the length of the target DNA sequence(s) can reduce false positives. Therefore, we present HisTrader, a computational tool to identify the regions accessible to transcription factors, nucleosome free regions (NFRs), within histone modification peaks to reduce the DNA sequence length required for motif analyses.ResultsHisTrader accurately identifies NFRs from H3K27Ac ChIP-seq profiles of the lung cancer cell line A549, which are validated by the presence of DNaseI hypersensitivity. In addition, HisTrader reveals that multiple NFRs are common within individual regulatory elements; an easily overlooked feature that should be considered to improve sensitivity of motif analyses using histone modification ChIP-seq data.Availability and implementationThe HisTrader script is open-source and available on GitHub (https://github.com/SvenBaileyLab/Histrader) under a GNU general public license (GPLv3). HisTrader is written in PERL and can be run on any platform with PERL installed.

scholarly journals Improved single-cell ATAC-seq reveals chromatin dynamics of in vitro corticogenesis

2019 ◽  
Author(s):  
Ryan M. Mulqueen ◽  
Brooke A. DeRosa ◽  
Casey A. Thornton ◽  
Zeynep Sayar ◽  
Kristof A. Torkenczy ◽  
...  
Keyword(s):  
Single Cell ◽  
Gene Networks ◽  
Regulatory Gene ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Chromatin Dynamics ◽  
Cell Library ◽  
Fate Decision

AbstractDevelopment is a complex process that requires the precise modulation of regulatory gene networks controlled through dynamic changes in the epigenome. Single-cell-omic technologies provide an avenue for understanding the mechanisms of these processes by capturing the progression of epigenetic cell states during the course of cellular differentiation using in vitro or in vivo models1. However, current single-cell epigenomic methods are limited in the information garnered per individual cell, which in turn limits their ability to measure chromatin dynamics and state shifts. Single-cell combinatorial indexing (sci-) has been applied as a strategy for identifying single-cell-omic originating libraries and removes the necessity of single-cell, single-compartment chemistry2. Here, we report an improved sci-assay for transposase accessible chromatin by sequencing (ATAC-seq), which utilizes the small molecule inhibitor Pitstop 2™ (scip-ATAC-seq)3. We demonstrate that these improvements, which theoretically could be applied to any in situ transposition method for single-cell library preparation, significantly increase the ability of transposase to enter the nucleus and generate highly complex single-cell libraries, without altering biological signal. We applied sci-ATAC-seq and scip-ATAC-seq to characterize the chromatin dynamics of developing forebrain-like organoids, an in vitro model of human corticogenesis4. Using these data, we characterized novel putative regulatory elements, compared the epigenome of the organoid model to human cortex data, generated a high-resolution pseudotemporal map of chromatin accessibility through differentiation, and measured epigenomic changes coinciding with a neurogenic fate decision point. Finally, we combined transcription factor motif accessibility with gene activity (GA) scores to directly observe the dynamics of complex regulatory programs that regulate neurogenesis through developmental pseudotime. Overall, scip-ATAC-seq increases information content per cell and bolsters the potential for future single-cell studies into complex developmental processes.

scholarly journals Live cell PNA labelling enables erasable fluorescence imaging of membrane proteins

2020 ◽  
Author(s):  
Georgina C. Gavins ◽  
Katharina Gröger ◽  
Michael D. Bartoschek ◽  
Philipp Wolf ◽  
Annette G. Beck-Sickinger ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Cho Cells ◽  
Fluorescent Dyes ◽  
Coiled Coil ◽  
Dna Nanotechnology ◽  
Nucleic Acid Hybridization ◽  
Landing Platform ◽  
Acid Protein ◽  
Reaction Proceeds ◽  
A Cell

AbstractDNA nanotechnology is an emerging field, which promises fascinating opportunities for the manipulation and imaging of proteins on a cell surface. The key to progress in the area is the ability to create the nucleic acid-protein junction in the context of living cells. Here we report a covalent labelling reaction, which installs a biostable peptide nucleic acid (PNA) tag. The reaction proceeds within minutes and is specific for proteins carrying a 2 kDa coiled coil peptide tag. Once installed the PNA label serves as a generic landing platform that enables the recruitment of fluorescent dyes via nucleic acid hybridization. We demonstrate the versatility of this approach by recruiting different fluorophores, assembling multiple fluorophores for increased brightness, and achieving reversible labelling by way of toehold mediated strand displacement. Additionally, we show that labelling can be carried out using two different coiled coil systems, with EGFR and ETBR, on both HEK293 and CHO cells. Finally, we apply the method to monitor internalization of EGFR on CHO cells.

scholarly journals Expanding detection windows for discriminating single nucleotide variants using rationally designed DNA equalizer probes

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Guan A. Wang ◽  
Xiaoyu Xie ◽  
Hayam Mansour ◽  
Fangfang Chen ◽  
Gabriela Matamoros ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Rural Areas ◽  
Simultaneous Detection ◽  
Dna Nanotechnology ◽  
Quantitative Relationship ◽  
Nucleic Acid Amplification ◽  
Nucleic Acid Hybridization ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Hybridization Probes

Abstract Combining experimental and simulation strategies to facilitate the design and operation of nucleic acid hybridization probes are highly important to both fundamental DNA nanotechnology and diverse biological/biomedical applications. Herein, we introduce a DNA equalizer gate (DEG) approach, a class of simulation-guided nucleic acid hybridization probes that drastically expand detection windows for discriminating single nucleotide variants in double-stranded DNA (dsDNA) via the user-definable transformation of the quantitative relationship between the detection signal and target concentrations. A thermodynamic-driven theoretical model was also developed, which quantitatively simulates and predicts the performance of DEG. The effectiveness of DEG for expanding detection windows and improving sequence selectivity was demonstrated both in silico and experimentally. As DEG acts directly on dsDNA, it is readily adaptable to nucleic acid amplification techniques, such as polymerase chain reaction (PCR). The practical usefulness of DEG was demonstrated through the simultaneous detection of infections and the screening of drug-resistance in clinical parasitic worm samples collected from rural areas of Honduras.

In Vitro Nucleic Acid Hybridization for the Identification of High-Risk Human Papillomavirus (HPV) in Cervical Clinical Specimens

2019 ◽  
Vol 42 ◽  
pp. 51-58
Author(s):  
N.A. Parmin ◽  
Uda Hashim ◽  
Wan Azani Mustafa ◽  
S.C.B. Gopinath ◽  
Zulida Rejali ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Risk ◽  
Test Specimen ◽  
Pap Smear ◽  
Nucleic Acid Hybridization ◽  
Clinical Specimens ◽  
Hpv Dna ◽  
Hpv Genotyping ◽  
High Risk Hpv

The reliability and specificity of the Pap smear rely on the eyewitness to perceive and an assortment of cell variations in clinically obtained cervical specimens. The improvement in early diagnosis to distinguish Human Papillomavirus (HPV) infection has been an issue. ELISA has been intended to analyze the immune response against HPV, and they can be utilized to distinguish the phase of the infection. The objective of this study to characterize the performance of In Vitro Nucleic Acid Hybridization with a correlation with Liquid-Based Cytology and HPV DNA Genotyping Test to assess its use as the potential for the identification of high-risk HPV in cervical clinical specimens. Hybridization utilizing an in vitro nucleic acid DNA-based ELISA method was performed with an improved chemiluminescent for the qualitative and semi-quantitative identification of high-risk HPV in cervical specimens. For analyzing the reliability to identify HPV DNA in cervical Pap smear, we studied the effectiveness of FDA-approved Hybrid Capture II (HCII) utilizing cervical swapping from 20 patient clinical specimens. Two scrapes-outs were positive for the occurrence of HPV. Only specimens displayed positive with HCII test were further confirmed by HPV Genotyping Test. Specimen 2 and 13 were positive for one of the 13 high-risk types in HCII test. Specimen 2 was confirmed by HPV genotyping test as a positive high-risk HPV type 58.

Abstract 611: AP-1 and ETS Family Transcription Factors Co-localize at Enhancers in Human Aortic Endothelial Cells

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Nicholas T Hogan ◽  
Casey E Romanoski ◽  
Michael T Lam ◽  
Christopher K Glass
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Transcription Factors ◽  
De Novo ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Motif Analysis ◽  
Aortic Endothelial Cells ◽  
Human Aortic Endothelial Cells ◽  
Aortic Endothelial

Introduction: Sequence-specific transcription factors bind DNA regulatory elements and play a key role in establishing cellular identity. Studies comparing macrophages to B cells have revealed that small numbers of such collaborative or lineage-determining transcription factors (LDTF) establish distinct enhancers in each cell type. These factors also allow for the binding of signal dependent transcription factors. Here we present data which suggest members of the AP-1, ETS, and STAT transcription factor families serve as collaborative transcriptional regulators in human aortic endothelial cells (HAEC). Hypothesis: We hypothesize that a set of AP-1 and ETS transcription factors collaborate to establish key endothelial cell enhancers. Methods: Working in HAEC, we measured poised and active enhancers using ChIP-seq for the epigenetic histone modifications H3K4me2 and H3K27Ac, performed motif analysis, and measured transcription factor binding for candidate factors. Knockdowns of JUN, ERG, and STAT3 followed by RNA-seq were used to evaluate altered enhancer function and gene targets of candidate factors. Results: Our de novo motif analysis revealed that motifs for ETS and AP-1 transcription factors are highly enriched at HAEC enhancers. ChIP-seq experiments for JUN, JUNB, ERG, and STAT3 showed between 8,000 and 55,000 intergenic peaks for each factor. Together these peaks bind 50% of poised enhancers, with a subset co-localizing at these sites. Gene ontology analysis showed that gene targets of these enhancers are involved in endothelial-specific functions. Further, knockdown of JUN, ERG, and STAT3 resulted in a twofold or greater change in expression of hundreds of HAEC transcripts. Conclusion: The genome-wide pattern of JUN, JUNB, ERG, and STAT3 co-localization at enhancers in HAEC suggests these factors serve as key regulators that collaboratively modulate endothelial-specific gene expression. Further investigation of candidate lineage-determining transcription factors using pro-atherogenic signals could reveal regulatory mechanisms of disease-relevant endothelial transcriptional programs.

Expanding the analytical applications of nucleic acid hybridization using junction probes

2020 ◽  
Vol 12 (41) ◽  
pp. 4931-4938
Author(s):  
Xuchu Wang ◽  
Zhihua Tao
Keyword(s):  
Nucleic Acid ◽  
Target Recognition ◽  
Nucleic Acid Hybridization ◽  
Analytical Applications

Nucleic acid hybridization is crucial in target recognition with respect to in vitro and in vivo nucleic acid biosensing.

scholarly journals Binding of the RING Polycomb Proteins to Specific Target Genes in Complex with the grainyhead-Like Family of Developmental Transcription Factors

2002 ◽  
Vol 22 (6) ◽  
pp. 1936-1946 ◽  
Author(s):  
Annabel Tuckfield ◽  
David R. Clouston ◽  
Tomasz M. Wilanowski ◽  
Lin-Lin Zhao ◽  
John M. Cunningham ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Target Genes ◽  
Regulatory Elements ◽  
Polycomb Group ◽  
Mobility Shift ◽  
Polycomb Proteins ◽  
Complex Forms ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The Polycomb group (PcG) of proteins represses homeotic gene expression through the assembly of multiprotein complexes on key regulatory elements. The mechanisms mediating complex assembly have remained enigmatic since most PcG proteins fail to bind DNA. We now demonstrate that the human PcG protein dinG interacts with CP2, a mammalian member of the grainyhead-like family of transcription factors, in vitro and in vivo. The functional consequence of this interaction is repression of CP2-dependent transcription. The CP2-dinG interaction is conserved in evolution with the Drosophila factor grainyhead binding to dring, the fly homologue of dinG. Electrophoretic mobility shift assays demonstrate that the grh-dring complex forms on regulatory elements of genes whose expression is repressed by grh but not on elements where grh plays an activator role. These observations reveal a novel mechanism by which PcG proteins may be anchored to specific regulatory elements in developmental genes.

Sign in / Sign up

Export Citation Format

Share Document