scholarly journals Massively parallel characterization of regulatory dynamics during neural induction

2018 ◽  
Author(s):  
Fumitaka Inoue ◽  
Anat Kreimer ◽  
Tal Ashuach ◽  
Nadav Ahituv ◽  
Nir Yosef
Keyword(s):  
Embryonic Stem ◽  
Neural Induction ◽  
Regulatory Elements ◽  
Massively Parallel ◽  
Computational Tools ◽  
Time Points ◽  
Genomic Assays ◽  
Prioritization Method ◽  
Reporter Assays ◽  
Molecular Components

AbstractThe molecular components governing neural induction remain largely unknown. Here, we applied a suite of genomic and computational tools to comprehensively identify these components. We performed RNA-seq, ChIP-seq (H3K27ac, H3K27me3) and ATAC-seq on human embryonic stem cells (hESCs) at seven early neural differentiation time points (0-72 hours) and identified thousands of induced genes and regulatory regions. We analyzed the function of ~2,500 selected regions using massively parallel reporter assays at all time points. We found numerous temporal enhancers that correlated with similarly timed epigenetic marks and gene expression. Development of a prioritization method that incorporated all genomic data identified key transcription factors (TFs) involved in neural induction. Individual overexpression of eleven TFs and several combinations in hESCs found novel neural induction regulators. Combined, our results provide a comprehensive map of genes and functional regulatory elements involved in neural induction and identify master regulator TFs that are instrumental for this process.One Sentence SummaryUsing numerous genomic assays and computational tools we characterized the dynamic changes that take place during neural induction.

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 Neural lineage induction reveals multi-scale dynamics of 3D chromatin organization

2014 ◽  
Author(s):  
Aleksandra Pekowska ◽  
Bernd Klaus ◽  
Felix Alexander Klein ◽  
Simon Anders ◽  
Malgorzata Oles ◽  
...  
Keyword(s):  
Gene Expression ◽  
Regulation Of Gene Expression ◽  
Embryonic Stem ◽  
Neural Induction ◽  
Interaction Network ◽  
Regulatory Elements ◽  
Chromatin Organization ◽  
Spatial Proximity ◽  
Enhancer Element ◽  
Neural Lineage

Regulation of gene expression underlies cell identity. Chromatin structure and gene activity are linked at multiple levels, via positioning of genomic loci to transcriptionally permissive or repressive environments and by connecting cis-regulatory elements such as promoters and enhancers. However, the genome-wide dynamics of these processes during cell differentiation has not been characterized. Using tethered chromatin conformation capture (TCC) sequencing we determined global three-dimensional chromatin structures in mouse embryonic stem (ES) and neural stem (NS) cell derivatives. We found that changes in the propensity of genomic regions to form inter-chromosomal contacts are pervasive in neural induction and are associated with the regulation of gene expression. Moreover, we found a pronounced contribution of euchromatic domains to the intra-chromosomal interaction network of pluripotent cells, indicating the existence of an ES cell-specific mode of chromatin organization. Mapping of promoter-enhancer interactions in pluripotent and differentiated cells revealed that spatial proximity without enhancer element activity is a common architectural feature in cells undergoing early developmental changes. Activity-independent formation of higher-order contacts between cis-regulatory elements, predominant at complex loci, may thus provide an additional layer of transcriptional control.

scholarly journals Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction

2019 ◽  
Vol 25 (5) ◽  
pp. 713-727.e10 ◽  
Author(s):  
Fumitaka Inoue ◽  
Anat Kreimer ◽  
Tal Ashuach ◽  
Nadav Ahituv ◽  
Nir Yosef
Keyword(s):  
Neural Induction ◽  
Regulatory Elements ◽  
Massively Parallel

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 Massively parallel dissection of human accelerated regions in human and chimpanzee neural progenitors

2018 ◽  
Author(s):  
Hane Ryu ◽  
Fumitaka Inoue ◽  
Sean Whalen ◽  
Alex Williams ◽  
Martin Kircher ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Neural Progenitors ◽  
Massively Parallel ◽  
Evolutionary Changes ◽  
Gene Regulatory Elements ◽  
Reporter Assays ◽  
Differentiation Stage ◽  
Almost All ◽  
Human Accelerated Regions ◽  
Human Specific

SUMMARYHow mutations in gene regulatory elements lead to evolutionary changes remains largely unknown. Human accelerated regions (HARs) are ideal for exploring this question, because they are associated with human-specific traits and contain multiple human-specific variants at sites conserved across mammals, suggesting that they alter or compensate to preserve function. We performed massively parallel reporter assays on all human and chimpanzee HAR sequences in human and chimpanzee iPSC-derived neural progenitors at two differentiation stages. Forty-three percent (306/714) of HARs function as neuronal enhancers, with two-thirds (204/306) showing consistent changes in activity between human and chimpanzee sequences. These changes were almost all sequence dependent and not affected by cell species or differentiation stage. We tested all evolutionary intermediates between human and chimpanzee sequences of seven HARs, finding variants that interact both positively and negatively. This study shows that variants acquired during human evolution interact to buffer and amplify changes to enhancer function.

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 Sequence-based correction of barcode bias in massively parallel reporter assays

2021 ◽  
Author(s):  
Dongwon Lee ◽  
Ashish Kapoor ◽  
Changhee Lee ◽  
Michael Mudgett ◽  
Michael A. Beer ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Dna Sequences ◽  
Regulatory Elements ◽  
Massively Parallel ◽  
Regulatory Variants ◽  
Specific Effects ◽  
High Throughput Method ◽  
Reporter Assays ◽  
Post Transcriptional Regulation

ABSTRACTMassively parallel reporter assays (MPRA) are a high-throughput method for evaluating in vitro activities of thousands of candidate cis-regulatory elements (CREs). In these assays, candidate sequences are cloned upstream or downstream of a reporter gene tagged by unique DNA sequences. However, tag sequences may themselves affect reporter gene expression and lead to major potential biases in the measured cis-regulatory activity. Here, we present a sequence-based method for correcting tag sequence-specific effects and demonstrate that our method can significantly reduce this source of variation, and improve the identification of functional regulatory variants by MPRAs. We also show that our model captures sequence features associated with post-transcriptional regulation of mRNA. Thus, this new method helps to not only improve detection of regulatory signals in MPRA experiments but also to design better MPRA protocols.

scholarly journals QuASAR-MPRA: Accurate allele-specific analysis for massively parallel reporter assays

2017 ◽  
Author(s):  
Cynthia A. Kalita ◽  
Gregory A. Moyerbrailean ◽  
Christopher Brown ◽  
Xiaoquan Wen ◽  
Francesca Luca ◽  
...  
Keyword(s):  
Data Analysis ◽  
High Throughput ◽  
Regulatory Elements ◽  
Massively Parallel ◽  
Regulatory Function ◽  
Regulatory Sequences ◽  
Analysis Tools ◽  
Specific Analysis ◽  
Allele Specific ◽  
Reporter Assays

ABSTRACTMotivationThe majority of the human genome is composed of non-coding regions containing regulatory elements such as enhancers, which are crucial for controlling gene expression. Many variants associated with complex traits are in these regions, and may disrupt gene regulatory sequences. Consequently, it is important to not only identify true enhancers but also to test if a variant within an enhancer affects gene regulation. Recently, allele-specific analysis in high-throughput reporter assays, such as massively parallel reporter assays (MPRA), have been used to functionally validate non-coding variants. However, we are still missing high-quality and robust data analysis tools for these datasets.ResultsWe have further developed our method for allele-specific analysis QuASAR (quantitative allele-specific analysis of reads) to analyze allele-specific signals in barcoded read counts data from MPRA. Using this approach, we can take into account the uncertainty on the original plasmid proportions, over-dispersion, and sequencing errors. The provided allelic skew estimate and its standard error also simplifies meta-analysis of replicate experiments. Additionally, we show that a beta-binomial distribution better models the variability present in the allelic imbalance of these synthetic reporters and results in a test that is statistically well calibrated under the null. Applying this approach to the MPRA data by Tewheyet al.(2016), we found 602 SNPs with significant (FDR 10%) allele-specific regulatory function in LCLs. We also show that we can combine MPRA with QuASAR estimates to validate existing experimental and computational annotations of regulatory variants. Our study shows that with appropriate data analysis tools, we can improve the power to detect allelic effects in high throughput reporter assays.Availabilityhttp://github.com/piquelab/QuASAR/tree/master/[email protected];[email protected]

scholarly journals Evolution of mouse circadian enhancers from transposable elements

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Julius Judd ◽  
Hayley Sanderson ◽  
Cédric Feschotte
Keyword(s):  
Gene Expression ◽  
Transposable Elements ◽  
Binding Sites ◽  
Mouse Liver ◽  
Integration Site ◽  
Point Mutations ◽  
Regulatory Elements ◽  
Circadian Gene ◽  
Binding Motifs ◽  
Reporter Assays

Abstract Background Transposable elements are increasingly recognized as a source of cis-regulatory variation. Previous studies have revealed that transposons are often bound by transcription factors and some have been co-opted into functional enhancers regulating host gene expression. However, the process by which transposons mature into complex regulatory elements, like enhancers, remains poorly understood. To investigate this process, we examined the contribution of transposons to the cis-regulatory network controlling circadian gene expression in the mouse liver, a well-characterized network serving an important physiological function. Results ChIP-seq analyses reveal that transposons and other repeats contribute ~ 14% of the binding sites for core circadian regulators (CRs) including BMAL1, CLOCK, PER1/2, and CRY1/2, in the mouse liver. RSINE1, an abundant murine-specific SINE, is the only transposon family enriched for CR binding sites across all datasets. Sequence analyses and reporter assays reveal that the circadian regulatory activity of RSINE1 stems from the presence of imperfect CR binding motifs in the ancestral RSINE1 sequence. These motifs matured into canonical motifs through point mutations after transposition. Furthermore, maturation occurred preferentially within elements inserted in the proximity of ancestral CR binding sites. RSINE1 also acquired motifs that recruit nuclear receptors known to cooperate with CRs to regulate circadian gene expression specifically in the liver. Conclusions Our results suggest that the birth of enhancers from transposons is predicated both by the sequence of the transposon and by the cis-regulatory landscape surrounding their genomic integration site.

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