scholarly journals Methods of massive parallel reporter assays for investigation of enhancers

2021 ◽  
Vol 25 (3) ◽  
pp. 344-355
Author(s):  
S. E. Romanov ◽  
D. A. Kalashnikova ◽  
P. P. Laktionov
Keyword(s):  
High Throughput ◽  
In Silico ◽  
Functional Role ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Regulatory Sequences ◽  
Gene Regulatory Elements ◽  
Experimental Approaches ◽  
Gene Regulatory

The correct deployment of genetic programs for development and differentiation relies on finely coordinated regulation of specific gene sets. Genomic regulatory elements play an exceptional role in this process. There are few types of gene regulatory elements, including promoters, enhancers, insulators and silencers. Alterations of gene regulatory elements may cause various pathologies, including cancer, congenital disorders and autoimmune diseases. The development of high-throughput genomic assays has made it possible to significantly accelerate the accumulation of information about the characteristic epigenetic properties of regulatory elements. In combination with high-throughput studies focused on the genome-wide distribution of epigenetic marks, regulatory proteins and the spatial structure of chromatin, this significantly expands the understanding of the principles of epigenetic regulation of genes and allows potential regulatory elements to be searched for in silico. However, common experimental approaches used to study the local characteristics of chromatin have a number of technical limitations that may reduce the reliability of computational identification of genomic regulatory sequences. Taking into account the variability of the functions of epigenetic determinants and complex multicomponent regulation of genomic elements activity, their functional verification is often required. A plethora of methods have been developed to study the functional role of regulatory elements on the genome scale. Common experimental approaches for in silico identification of regulatory elements and their inherent technical limitations will be described. The present review is focused on original high-throughput methods of enhancer activity reporter analysis that are currently used to validate predicted regulatory elements and to perform de novo searches. The methods described allow assessing the functional role of the nucleotide sequence of a regulatory element, to determine its exact boundaries and to assess the influence of the local state of chromatin on the activity of enhancers and gene expression. These approaches have contributed substantially to the understanding of the fundamental principles of gene regulation.

scholarly journals Remotely acting SMCHD1 gene regulatory elements: in silico prediction and identification of potential regulatory variants in patients with FSHD

2015 ◽  
Vol 9 (1) ◽  
Author(s):  
Mary B. Mayes ◽  
Taniesha Morgan ◽  
Jincy Winston ◽  
Daniel S. Buxton ◽  
Mihir Anant Kamat ◽  
...  
Keyword(s):  
In Silico ◽  
Regulatory Elements ◽  
In Silico Prediction ◽  
Regulatory Variants ◽  
Gene Regulatory Elements ◽  
Gene Regulatory

A High Throughput Assay of Lichenase Activity with Congo Red Dye in Plants

2021 ◽  
Author(s):  
Alexander Tyurin ◽  
Aleksandra V. Suhorukova ◽  
Igor V. Deineko ◽  
Olga S. Pavlenko ◽  
Viktoriia A. Fridman ◽  
...  
Keyword(s):  
Plant Cell ◽  
High Throughput ◽  
Experimental Verification ◽  
Congo Red ◽  
Functional Role ◽  
Specific Interaction ◽  
Regulatory Sequences ◽  
Reporter Protein ◽  
Verification Methods

Abstract Background : To elucidate the functional role of regulatory sequences and contexts identified and predicted during omics studies in the complex mechanisms of gene expression regulation, experimental verification methods in a plant cell are required. For this, the method of transient expression of reporter genes fused with the tested sequences is widely used. Several reporter systems are available that have shown good performance in the studies including the thermostable lichenase Clostridium thermocellum . However, each reporter system has its own limitations with respect to the quantification of the protein product of a reporter gene and, in particular, for the use of high-throughput approaches. Results: In this study, we report the design a high throughput fluorometric method for quantification of thermostable lichenase C. thermocellum using Congo red and further experimental verification of its relevance and efficiency in assessment of the functional role of regulatory sequences in the plant cell. Conclusion: The specific interaction between the dye Congo red and β -D-glucans formed the background for designing a high-throughput fluorometric assay for quantification of C. thermocellum thermostable lichenase as a reporter protein for plants. This assay (i) makes it possible to precisely measure the amount of reporter protein in a plant sample; (ii) has shown a high sensitivity for quantification of thermostable lichenase; (iii) is more time- and cost-efficient as compared with the Somogyi–Nelson assay; and (iv) is to the least degree dependent on the presence of the tested buffer components as compared with the Somogyi–Nelson assay.

scholarly journals Screening in silico predicted remotely acting NF1 gene regulatory elements for mutations in patients with neurofibromatosis type 1

2013 ◽  
Vol 7 (1) ◽  
pp. 18 ◽  
Author(s):  
Stephen E Hamby ◽  
Pablo Reviriego ◽  
David N Cooper ◽  
Meena Upadhyaya ◽  
Nadia Chuzhanova
Keyword(s):  
Neurofibromatosis Type 1 ◽  
In Silico ◽  
Neurofibromatosis Type ◽  
Regulatory Elements ◽  
Nf1 Gene ◽  
Gene Regulatory Elements ◽  
Gene Regulatory

scholarly journals Predicting Gene Regulatory Elements in Silico on a Genomic Scale

1998 ◽  
Vol 8 (11) ◽  
pp. 1202-1215 ◽  
Author(s):  
Alvis Brāzma ◽  
Inge Jonassen ◽  
Jaak Vilo ◽  
Esko Ukkonen
Keyword(s):  
In Silico ◽  
Regulatory Elements ◽  
Gene Regulatory Elements ◽  
Genomic Scale ◽  
Gene Regulatory

scholarly journals 3D Chromatin Architecture Remodeling during Human Cardiomyocyte Differentiation Reveals A Role Of HERV-H In Demarcating Chromatin Domains

2018 ◽  
Author(s):  
Yanxiao Zhang ◽  
Ting Li ◽  
Sebastian Preissl ◽  
Jonathan Grinstein ◽  
Elie N. Farah ◽  
...  
Keyword(s):  
Regulatory Networks ◽  
De Novo ◽  
Heart Diseases ◽  
Regulatory Elements ◽  
Chromatin Domain ◽  
Specific Gene ◽  
Cardiomyocyte Differentiation ◽  
Chromatin Architecture ◽  
Gene Regulatory

AbstractDynamic restructuring of chromatin architecture has been implicated in cell-type specific gene regulatory programs; yet, how chromatin remodels during lineage specification remains to be elucidated. Through interrogating chromatin reorganization during human cardiomyocyte differentiation, we uncover dynamic chromatin interactions between genes and distal regulatory elements harboring noncoding variants associated with adult and congenital heart diseases. Unexpectedly, we also discover a new class of human pluripotent stem cell (PSC)-specific topologically associating domains (TAD) that are created by the actively transcribed endogenous retrotransposon HERV-H. Deletion or silencing of specific HERV-H elements eliminates corresponding TAD boundaries, while de novo insertion of HERV-H can introduce new chromatin domain boundaries in human PSCs. Furthermore, comparative analysis of chromatin architecture in other species that lack HERV-H sequences supports a role for actively transcribed HERV-H in demarcating human PSC-specific TADs. The biological role of HERV-H is further underscored by the observation that deletion of a specific HERV-H reduces transcription of genes upstream and facilitates cell differentiation. Overall, our results highlight a previously unrecognized role for retrotransposons in restructuring genome architecture in the human genome and delineate dynamic gene regulatory networks during cardiomyocyte development that inform how non-coding genetic variants contribute to human heart diseases.

scholarly journals lentiMPRA and MPRAflow for high-throughput functional characterization of gene regulatory elements

2020 ◽  
Vol 15 (8) ◽  
pp. 2387-2412 ◽  
Author(s):  
M. Grace Gordon ◽  
Fumitaka Inoue ◽  
Beth Martin ◽  
Max Schubach ◽  
Vikram Agarwal ◽  
...  
Keyword(s):  
High Throughput ◽  
Functional Characterization ◽  
Regulatory Elements ◽  
Gene Regulatory Elements ◽  
Gene Regulatory

scholarly journals Methylation Patterns and Chromatin Accessibility in Neuroendocrine Lung Cancer

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2003
Author(s):  
Elsa Arbajian ◽  
Mattias Aine ◽  
Anna Karlsson ◽  
Johan Vallon-Christersson ◽  
Hans Brunnström ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Lines ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Specific Gene ◽  
Rna Seq ◽  
Lung Cancers ◽  
Gene Regulatory Elements ◽  
Gene Regulatory ◽  
Methylation Patterns

Lung cancer is the worldwide leading cause of death from cancer. Epigenetic modifications such as methylation and changes in chromatin accessibility are major gene regulatory mechanisms involved in tumorigenesis and cellular lineage commitment. We aimed to characterize these processes in the context of neuroendocrine (NE) lung cancer. Illumina 450K DNA methylation data were collected for 1407 lung cancers including 27 NE tumors. NE differentially methylated regions (NE-DMRs) were identified and correlated with gene expression data for 151 lung cancers and 31 human tissue entities from the Genotype-Tissue Expression (GTEx) consortium. Assay for transposase-accessible chromatin sequencing (ATAC-seq) and RNA sequencing (RNA-seq) were performed on eight lung cancer cell lines, including three NE cell lines, to identify neuroendocrine specific gene regulatory elements. We identified DMRs with methylation patterns associated with differential gene expression and an NE tumor phenotype. DMR-associated genes could further be split into six functional modules, including one highly specific gene module for NE lung cancer showing high expression in both normal and malignant brain tissue. The regulatory potential of NE-DMRs was further validated in vitro using paired ATAC- and RNA-seq and revealed both proximal and distal regulatory elements of canonical NE-marker genes such as CHGA, NCAM1, INSM1, as well as a number of novel candidate markers of NE lung cancer. Using multilevel genomic analyses of both tumor bulk tissue and lung cancer cell lines, we identified a large catalogue of gene regulatory elements related to the NE phenotype of lung cancer.

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