scholarly journals TransMPRA: A framework for assaying the role of many trans-acting factors at many enhancers

2020 ◽  
Author(s):  
Diego Calderon ◽  
Andria Ellis ◽  
Riza M. Daza ◽  
Beth Martin ◽  
Jacob M. Tome ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Transcription Factors ◽  
Regulatory Elements ◽  
Massively Parallel ◽  
Reporter Assay ◽  
Regulatory Activity ◽  
Large Numbers ◽  
Reporter Assays ◽  
Regulatory Potential

AbstractGene regulation occurs through trans-acting factors (e.g. transcription factors) acting on cis-regulatory elements (e.g. enhancers). Massively parallel reporter assays (MPRAs) functionally survey large numbers of cis-regulatory elements for regulatory potential, but do not identify the trans-acting factors that mediate any observed effects. Here we describe transMPRA — a reporter assay that efficiently combines multiplex CRISPR-mediated perturbation and MPRAs to identify trans-acting factors that modulate the regulatory activity of specific enhancers.

scholarly journals A systematic evaluation of the design, orientation, and sequence context dependencies of massively parallel reporter assays

2019 ◽  
Author(s):  
Jason Klein ◽  
Vikram Agarwal ◽  
Fumitaka Inoue ◽  
Aidan Keith ◽  
Beth Martin ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Experimental Design ◽  
Massively Parallel ◽  
Systematic Evaluation ◽  
Regulatory Activity ◽  
Sequence Context ◽  
Enhancer Activity ◽  
Extended Sequence ◽  
Reporter Assays ◽  
Novel Method

ABSTRACTMassively parallel reporter assays (MPRAs) functionally screen thousands of sequences for regulatory activity in parallel. Although MPRAs have been applied to address diverse questions in gene regulation, there has been no systematic comparison of how differences in experimental design influence findings. Here, we screen a library of 2,440 sequences, representing candidate liver enhancers and controls, in HepG2 cells for regulatory activity using nine different approaches (including conventional episomal, STARR-seq, and lentiviral MPRA designs). We identify subtle but significant differences in the resulting measurements that correlate with epigenetic and sequence-level features. We also test this library in both orientations with respect to the promoter, validatingen massethat enhancer activity is robustly independent of orientation. Finally, we develop and apply a novel method to assemble and functionally test libraries of the same putative enhancers as 192-mers, 354-mers, and 678-mers, and observe surprisingly large differences in functional activity. This work provides a framework for the experimental design of high-throughput reporter assays, suggesting that the extended sequence context of tested elements, and to a lesser degree the precise assay, influence MPRA results.

scholarly journals MPRAnator: a web-based tool for the design of Massively Parallel Reporter Assay experiments.

2015 ◽  
Author(s):  
Ilias Georgakopoulos-Soares ◽  
Naman Jain ◽  
Jesse Gray ◽  
Martin Hemberg
Keyword(s):  
High Throughput ◽  
Regulatory Elements ◽  
Massively Parallel ◽  
Regulatory Sequences ◽  
Reporter Assay ◽  
Sequencing Technologies ◽  
Fine Control ◽  
Reporter Assays ◽  
Dna Regulatory Elements ◽  
Massively Parallel Reporter Assay

DNA regulatory elements contain short motifs where transcription factors (TFs) can bind to modulate gene expression. Although the broad principles of TF regulation are well understood, the rules that dictate how combinatorial TF binding translates into transcriptional activity remain largely unknown. With the rapid advances in DNA synthesis and sequencing technologies and the continuing decline in the associated costs, high-throughput experiments can be performed to investigate the regulatory role of thousands of oligonucleotide sequences simultaneously. Nevertheless, designing high-throughput reporter assay experiments such as Massively Parallel Reporter Assays (MPRAs) and similar methods remains challenging. We introduce MPRAnator, a set of tools that facilitate rapid design of MPRA experiments. With MPRA Motif design, a set of variables provides fine control of how motifs are placed into sequences therefore allowing the user to investigate the rules that govern TF occupancy. MPRA SNP design can be used to investigate the functional effects of single or combinations of SNPs at regulatory sequences. Finally, the Transmutation tool allows for the design of negative controls by permitting scrambling, reversing, complementing or introducing multiple random mutations in the input sequences or motifs.

scholarly journals Identification of long regulatory elements in the genome of Plasmodium falciparum and other eukaryotes

2021 ◽  
Vol 17 (4) ◽  
pp. e1008909
Author(s):  
Christophe Menichelli ◽  
Vincent Guitard ◽  
Rafael M. Martins ◽  
Sophie Lèbre ◽  
Jose-Juan Lopez-Rubio ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Transcription Factors ◽  
Binding Sites ◽  
Cpg Islands ◽  
Regulatory Elements ◽  
Reporter Assay ◽  
Development Stages ◽  
Multicellular Organisms ◽  
Post Transcriptional Regulation

Long regulatory elements (LREs), such as CpG islands, polydA:dT tracts or AU-rich elements, are thought to play key roles in gene regulation but, as opposed to conventional binding sites of transcription factors, few methods have been proposed to formally and automatically characterize them. We present here a computational approach named DExTER (Domain Exploration To Explain gene Regulation) dedicated to the identification of candidate LREs (cLREs) and apply it to the analysis of the genomes of P. falciparum and other eukaryotes. Our analyses show that all tested genomes contain several cLREs that are somewhat conserved along evolution, and that gene expression can be predicted with surprising accuracy on the basis of these long regions only. Regulation by cLREs exhibits very different behaviours depending on species and conditions. In P. falciparum and other Apicomplexan organisms as well as in Dictyostelium discoideum, the process appears highly dynamic, with different cLREs involved at different phases of the life cycle. For multicellular organisms, the same cLREs are involved in all tissues, but a dynamic behavior is observed along embryonic development stages. In P. falciparum, whose genome is known to be strongly depleted of transcription factors, cLREs are predictive of expression with an accuracy above 70%, and our analyses show that they are associated with both transcriptional and post-transcriptional regulation signals. Moreover, we assessed the biological relevance of one LRE discovered by DExTER in P. falciparum using an in vivo reporter assay. The source code (python) of DExTER is available at https://gite.lirmm.fr/menichelli/DExTER.

scholarly journals Epigenomic Analysis of RAD51 ChIP-seq Data Reveals cis-regulatory Elements Associated with Autophagy in Cancer Cell Lines

Cancers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2547
Author(s):  
Keunsoo Kang ◽  
Yoonjung Choi ◽  
Hyeonjin Moon ◽  
Chaelin You ◽  
Minjin Seo ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Homologous Recombination ◽  
Cell Lines ◽  
Regulatory Elements ◽  
Binding Motif ◽  
Genome Wide ◽  
E Box ◽  
Autophagy Pathway ◽  
Mcf 7

RAD51 is a recombinase that plays a pivotal role in homologous recombination. Although the role of RAD51 in homologous recombination has been extensively studied, it is unclear whether RAD51 can be involved in gene regulation as a co-factor. In this study, we found evidence that RAD51 may contribute to the regulation of genes involved in the autophagy pathway with E-box proteins such as USF1, USF2, and/or MITF in GM12878, HepG2, K562, and MCF-7 cell lines. The canonical USF binding motif (CACGTG) was significantly identified at RAD51-bound cis-regulatory elements in all four cell lines. In addition, genome-wide USF1, USF2, and/or MITF-binding regions significantly coincided with the RAD51-associated cis-regulatory elements in the same cell line. Interestingly, the promoters of genes associated with the autophagy pathway, such as ATG3 and ATG5, were significantly occupied by RAD51 and regulated by RAD51 in HepG2 and MCF-7 cell lines. Taken together, these results unveiled a novel role of RAD51 and provided evidence that RAD51-associated cis-regulatory elements could possibly be involved in regulating autophagy-related genes with E-box binding proteins.

scholarly journals sRNA/L1 retrotransposition: using siRNAs and miRNAs to expand the applications of the cell culture-based LINE-1 retrotransposition assay

2020 ◽  
Vol 375 (1795) ◽  
pp. 20190346 ◽  
Author(s):  
Pablo Tristan-Ramos ◽  
Santiago Morell ◽  
Laura Sanchez ◽  
Belen Toledo ◽  
Jose L. Garcia-Perez ◽  
...  
Keyword(s):  
Cell Culture ◽  
Gene Regulation ◽  
Small Rna ◽  
Host Factors ◽  
Fanconi Anaemia ◽  
Reporter Assay ◽  
Long Interspersed Elements ◽  
Essential Tool ◽  
Conventional Cell

The cell culture-based retrotransposition reporter assay has been (and is) an essential tool for the study of vertebrate Long INterspersed Elements (LINEs). Developed more than 20 years ago, this assay has been instrumental in characterizing the role of LINE-encoded proteins in retrotransposition, understanding how ribonucleoprotein particles are formed, how host factors regulate LINE mobilization, etc. Moreover, variations of the conventional assay have been developed to investigate the biology of other currently active human retrotransposons, such as Alu and SVA. Here, we describe a protocol that allows combination of the conventional cell culture-based LINE-1 retrotransposition reporter assay with short interfering RNAs (siRNAs) and microRNA (miRNAs) mimics or inhibitors, which has allowed us to uncover specific miRNAs and host factors that regulate retrotransposition. The protocol described here is highly reproducible, quantitative, robust and flexible, and allows the study of several small RNA classes and various retrotransposons. To illustrate its utility, here we show that siRNAs to Fanconi anaemia proteins (FANC-A and FANC-C) and an inhibitor of miRNA-20 upregulate and downregulate human L1 retrotransposition, respectively. This article is part of a discussion meeting issue ‘Crossroads between transposons and gene regulation’.

scholarly journals Transposable elements as a potent source of diverse cis -regulatory sequences in mammalian genomes

2020 ◽  
Vol 375 (1795) ◽  
pp. 20190347 ◽  
Author(s):  
Vasavi Sundaram ◽  
Joanna Wysocka
Keyword(s):  
Gene Regulation ◽  
Transposable Elements ◽  
Regulatory Networks ◽  
Regulatory Elements ◽  
Regulatory Function ◽  
Specific Gene ◽  
Regulatory Sequences ◽  
Dependent Manner ◽  
Unique Contribution ◽  
Regulatory Potential

Eukaryotic gene regulation is mediated by cis -regulatory elements, which are embedded within the vast non-coding genomic space and recognized by the transcription factors in a sequence- and context-dependent manner. A large proportion of eukaryotic genomes, including at least half of the human genome, are composed of transposable elements (TEs), which in their ancestral form carried their own cis -regulatory sequences able to exploit the host trans environment to promote TE transcription and facilitate transposition. Although not all present-day TE copies have retained this regulatory function, the preexisting regulatory potential of TEs can provide a rich source of cis -regulatory innovation for the host. Here, we review recent evidence documenting diverse contributions of TE sequences to gene regulation by functioning as enhancers, promoters, silencers and boundary elements. We discuss how TE-derived enhancer sequences can rapidly facilitate changes in existing gene regulatory networks and mediate species- and cell-type-specific regulatory innovations, and we postulate a unique contribution of TEs to species-specific gene expression divergence in pluripotency and early embryogenesis. With advances in genome-wide technologies and analyses, systematic investigation of TEs' cis -regulatory potential is now possible and our understanding of the biological impact of genomic TEs is increasing. This article is part of a discussion meeting issue ‘Crossroads between transposons and gene regulation’.

Role of AP2/EREBP transcription factors in gene regulation during abiotic stress

FEBS Letters ◽  
2001 ◽  
Vol 498 (2-3) ◽  
pp. 187-189 ◽  
Author(s):  
Dimosthenis Kizis ◽  
Victoria Lumbreras ◽  
Montserrat Pagès
Keyword(s):  
Gene Regulation ◽  
Transcription Factors ◽  
Abiotic Stress

scholarly journals Deciphering regulatory DNA sequences and noncoding genetic variants using neural network models of massively parallel reporter assays

2018 ◽  
Author(s):  
Rajiv Movva ◽  
Peyton Greenside ◽  
Georgi K. Marinov ◽  
Surag Nair ◽  
Avanti Shrikumar ◽  
...  
Keyword(s):  
Neural Network ◽  
Dna Sequences ◽  
Genetic Variants ◽  
Massively Parallel ◽  
Regulatory Sequence ◽  
Regulatory Activity ◽  
Neural Network Models ◽  
Regulatory Dna Sequences ◽  
Reporter Assays ◽  
Regulatory Dna

AbstractThe relationship between noncoding DNA sequence and gene expression is not well-understood. Massively parallel reporter assays (MPRAs), which quantify the regulatory activity of large libraries of DNA sequences in parallel, are a powerful approach to characterize this relationship. We present MPRA-DragoNN, a convolutional neural network (CNN)-based framework to predict and interpret the regulatory activity of DNA sequences as measured by MPRAs. While our method is generally applicable to a variety of MPRA designs, here we trained our model on the Sharpr-MPRA dataset that measures the activity of ~500,000 constructs tiling 15,720 regulatory regions in human K562 and HepG2 cell lines. MPRA-DragoNN predictions were moderately correlated (Spearman ρ = 0.28) with measured activity and were within range of replicate concordance of the assay. State-of-the-art model interpretation methods revealed high-resolution predictive regulatory sequence features that overlapped transcription factor (TF) binding motifs. We used the model to investigate the cell type and chromatin state preferences of predictive TF motifs. We explored the ability of our model to predict the allelic effects of regulatory variants in an independent MPRA experiment and fine map putative functional SNPs in loci associated with lipid traits. Our results suggest that interpretable deep learning models trained on MPRA data have the potential to reveal meaningful patterns in regulatory DNA sequences and prioritize regulatory genetic variants, especially as larger, higher-quality datasets are produced.

scholarly journals Massively parallel reporter perturbation assay uncovers temporal regulatory architecture during neural differentiation

2021 ◽  
Author(s):  
Anat Kreimer ◽  
Tal Ashuach ◽  
Fumitaka Inoue ◽  
Alex Khodaverdian ◽  
Nir Yosef ◽  
...  
Keyword(s):  
Neural Differentiation ◽  
Cellular Differentiation ◽  
Early Time ◽  
Regulatory Elements ◽  
Fine Tuning ◽  
Massively Parallel ◽  
Regulatory Architecture ◽  
Cellular Environment ◽  
Sequence Components ◽  
Reporter Assays

AbstractGene regulatory elements play a key role in orchestrating gene expression during cellular differentiation, but what determines their function over time remains largely unknown. Here, we performed perturbation-based massively parallel reporter assays at seven early time points of neural differentiation to systematically characterize how regulatory elements and motifs within them guide cellular differentiation. By perturbing over 2,000 putative DNA binding motifs in active regulatory regions, we delineated four categories of functional elements, and observed that activity direction is mostly determined by the sequence itself, while the magnitude of effect depends on the cellular environment. We also find that fine-tuning transcription rates is often achieved by a combined activity of adjacent activating and repressing elements. Our work provides a blueprint for the sequence components needed to induce different transcriptional patterns in general and specifically during neural differentiation.

scholarly journals Genome-Wide Analysis Reveals the Potential Role of MYB Transcription Factors in Floral Scent Formation in Hedychium coronarium

2021 ◽  
Vol 12 ◽  
Author(s):  
Farhat Abbas ◽  
Yanguo Ke ◽  
Yiwei Zhou ◽  
Yunyi Yu ◽  
Muhammad Waseem ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Gene Family ◽  
Floral Scent ◽  
Expression Patterns ◽  
Acid Methyl Ester ◽  
Regulatory Elements ◽  
Composition Analysis ◽  
Myb Gene ◽  
Hedychium Coronarium

The MYB gene family is one of the largest groups of transcription factors (TFs) playing diverse roles in several biological processes. Hedychium coronarium (white ginger lily) is a renowned ornamental plant both in tropical and subtropical regions due to its flower shape and strong floral scent mainly composed of terpenes and benzenoids. However, there is no information available regarding the role of the MYB gene family in H. coronarium. In the current study, the MYB gene family was identified and extensively analyzed. The identified 253 HcMYB genes were unevenly mapped on 17 chromosomes at a different density. Promoter sequence analysis showed numerous phytohormones related to cis-regulatory elements. The majority of HcMYB genes contain two to three introns and motif composition analysis showed their functional conservation. Phylogenetic analysis revealed that HcMYBs could be classified into 15 distinct clades, and the segmental duplication events played an essential role in the expansion of the HcMYB gene family. Tissue-specific expression patterns of HcMYB genes displayed spatial and temporal expression. Furthermore, seven HcMYB (HcMYB7/8/75/79/145/238/248) were selected for further investigation. Through RT-qPCR, the response of candidates HcMYB genes toward jasmonic acid methyl ester (MeJA), abscisic acid (ABA), ethylene, and auxin was examined. Yeast one-hybrid (Y1H) assays revealed that candidate genes directly bind to the promoter of bottom structural volatile synthesis genes (HcTPS1, HcTPS3, HcTPS10, and HcBSMT2). Moreover, yeast two-hybrid (Y2H) assay showed that HcMYB7/8/75/145/248 interact with HcJAZ1 protein. In HcMYB7/8/79/145/248-silenced flowers, the floral volatile contents were decreased and downregulated the expression of key structural genes, suggesting that these genes might play crucial roles in floral scent formation in H. coronarium by regulating the expression of floral scent biosynthesis genes. Collectively, these findings indicate that HcMYB genes might be involved in the regulatory mechanism of terpenoids and benzenoid biosynthesis in H. coronarium.

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