scholarly journals Identification of determinants of differential chromatin accessibility through a massively parallel genome-integrated reporter assay

2020 ◽  
Vol 30 (10) ◽  
pp. 1468-1480
Author(s):  
Jennifer Hammelman ◽  
Konstantin Krismer ◽  
Budhaditya Banerjee ◽  
David K. Gifford ◽  
Richard I. Sherwood
Keyword(s):  
Chromatin Accessibility ◽  
Massively Parallel ◽  
Reporter Assay

Abstract 18374: Targeted Sequencing and Massively Parallel Reporter Assay Identify the Functional Variation Underlying the 4q25 Locus for Atrial Fibrillation

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Nathan R Tucker ◽  
Jiangchuan Ye ◽  
Honghuang Lin ◽  
Michael A McLellan ◽  
Emelia J Benjamin ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Association Studies ◽  
Targeted Sequencing ◽  
Massively Parallel ◽  
Genome Wide Association Studies ◽  
Sequencing Analysis ◽  
Reporter Assay ◽  
Enhancer Activity ◽  
Functional Variants ◽  
Massively Parallel Reporter Assay

Introduction: Genome-wide association studies have identified 14 independent loci for atrial fibrillation (AF). The 4q25 locus upstream of the left-right asymmetry gene PITX2 is, by far, the strongest association signal for AF. However, as with most GWAS loci, the functional variants are noncoding, presumed to be regulatory, and remain unknown. We therefore sought to rapidly identify the functional variants at an AF locus by combining high throughput sequencing and massively parallel reporter assays. Methods and Results: We sequenced a ~750kb region encompassing the PITX2 locus in 462 individuals with early-onset AF from the MGH AF Study and 464 referents from the Framingham Heart Study. The SNP most significantly associated with AF in our sequenced sample was rs2129983, which is 140kb from PITX2 (OR=2.43, P =8.9X10 -16 ). rs2129983 is approximately 1.7kb from the most significantly associated SNP in a prior AF GWAS, rs6817105 (r 2 =0.52). From the targeted sequencing analysis, we identified 262 SNVs with a MAF >0.5% within a genomic region bounded by SNPs with an r2 greater than 0.4 with the top variant. To identify functional variants, we then utilized a massively parallel reporter assay (MPRA) in order to measure enhancer activity at each SNP across the entire AF locus. In both HL-1 and C2C12 myoblasts, MPRA identified many distinct SNP regions with differential enhancer activity. Using AF-association status as a standard, we were able to identify a series of variants that have both differential activity in either cell line tested and also a high level of association (rs17042076, rs4469143). Mechanistically, these functional SNPs are predicted to alter transcription factor binding. Conclusions: We have comprehensively identified the AF-associated variation at 4q25 and determined which of these variants are functional through differential enhancer activity. Here, in addition to identifying the causative variation for AF at 4q25, we provide a generalizable pathway for translating this work to other loci, a method that could expedite the identification of causative genetic variants at other disease loci.

scholarly journals A Human Accelerated Region is a Leydig cell GLI2 Enhancer that Affects Male-Typical Behavior

2021 ◽  
Author(s):  
Andrew R. Norman ◽  
Ann H. Ryu ◽  
Kirsty Jamieson ◽  
Sean Thomas ◽  
Yin Shen ◽  
...  
Keyword(s):  
Cell Line ◽  
Reproductive Biology ◽  
Leydig Cells ◽  
Massively Parallel ◽  
Reporter Assay ◽  
Enhancer Activity ◽  
Human Male ◽  
Enhancer Function ◽  
Massively Parallel Reporter Assay ◽  
Human Accelerated Regions

ABSTRACTHuman accelerated regions (HARs) are sequences that have evolved at an accelerated rate in the human lineage. Some HARs are developmental enhancers. We used a massively parallel reporter assay (MPRA) to identify HARs with enhancer activity in a mammalian testis cell line. A subset of HARs exhibited differential activity between the human and chimpanzee orthologs, representing candidates for underlying unique human male reproductive biology. We further characterized one of these candidate testis enhancers, 2xHAR.238. CRISPR/Cas9-mediated deletion in a testis cell line and mice revealed that 2xHAR.238 enhances expression of Gli2, encoding a Hedgehog pathway effector, in testis Leydig cells. 4C-seq revealed that 2xHAR.238 contacts the Gli2 promoter, consistent with enhancer function. In adult male mice, deletion of 2xHAR.238 disrupted mouse male-typical behavior and male interest in female odor. Combined, our work identifies a HAR that promotes the expression of Gli2 in Leydig cells and may have contributed to the evolution of human male reproductive biology.

scholarly journals High-throughput identification of RNA nuclear enrichment sequences

2017 ◽  
Author(s):  
Chinmay J Shukla ◽  
Alexandra L McCorkindale ◽  
Chiara Gerhardinger ◽  
Keegan D Korthauer ◽  
Moran N Cabili ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Nuclear Localization ◽  
Single Molecule ◽  
Noncoding Rnas ◽  
Massively Parallel ◽  
Reporter Assay ◽  
Sequence Elements ◽  
Massively Parallel Reporter Assay ◽  
Insight Into

SummaryOne of the biggest surprises since the sequencing of the human genome has been the discovery of thousands of long noncoding RNAs (lncRNAs)1–6. Although lncRNAs and mRNAs are similar in many ways, they differ with lncRNAs being more nuclear-enriched and in several cases exclusively nuclear7,8. Yet, the RNA-based sequences that determine nuclear localization remain poorly understood9–11. Towards the goal of systematically dissecting the lncRNA sequences that impart nuclear localization, we developed a massively parallel reporter assay (MPRA). Unlike previous MPRAs12–15 that determine motifs important for transcriptional regulation, we have modified this approach to identify sequences sufficient for RNA nuclear enrichment for 38 human lncRNAs. Using this approach, we identified 109 unique, conserved nuclear enrichment regions, originating from 29 distinct lncRNAs. We also discovered two shorter motifs within our nuclear enrichment regions. We further validated the sufficiency of several regions to impart nuclear localization by single molecule RNA fluorescence in situ hybridization (smRNA-FISH). Taken together, these results provide a first systematic insight into the sequence elements responsible for the nuclear enrichment of lncRNA molecules.

scholarly journals Systematic dissection of regulatory motifs in 2000 predicted human enhancers using a massively parallel reporter assay

2013 ◽  
Vol 23 (5) ◽  
pp. 800-811 ◽  
Author(s):  
P. Kheradpour ◽  
J. Ernst ◽  
A. Melnikov ◽  
P. Rogov ◽  
L. Wang ◽  
...  
Keyword(s):  
Massively Parallel ◽  
Reporter Assay ◽  
Regulatory Motifs ◽  
Massively Parallel Reporter Assay

SA131A MASSIVELY PARALLEL REPORTER ASSAY FOR VARIANTS ASSOCIATED WITH SCHIZOPHRENIA AND ALZHEIMER'S DISEASE

2019 ◽  
Vol 29 ◽  
pp. S1260-S1261
Author(s):  
Dimitrios Avramopoulos ◽  
Leslie Myint ◽  
Kasper Hansen ◽  
Ruihua Wang ◽  
Leandros Boukas ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Massively Parallel ◽  
Reporter Assay ◽  
Massively Parallel Reporter Assay

scholarly journals Development of a massively parallel reporter assay to identify functional regulatory variants in the PICALM Alzheimer disease associated locus

2020 ◽  
Vol 16 (S2) ◽  
Author(s):  
Karen Nuytemans ◽  
Derek Van Booven ◽  
Derek M. Dykxhoorn ◽  
Jeffery M. Vance ◽  
Margaret A. Pericak‐Vance ◽  
...  
Keyword(s):  
Alzheimer Disease ◽  
Massively Parallel ◽  
Reporter Assay ◽  
Regulatory Variants ◽  
Massively Parallel Reporter Assay

scholarly journals Massively parallel analysis of human 3′ UTRs reveals that AU-rich element length and registration predict mRNA destabilization

2021 ◽  
Author(s):  
David A Siegel ◽  
Olivier Le Tonqueze ◽  
Anne Biton ◽  
Noah Zaitlen ◽  
David J Erle
Keyword(s):  
Gene Expression ◽  
Mrna Stability ◽  
Regulatory Elements ◽  
Massively Parallel ◽  
Reporter Assay ◽  
Stem Loop ◽  
Mrna Destabilization ◽  
The Stability ◽  
Massively Parallel Reporter Assay ◽  
Simple Sequence

Abstract AU-rich elements (AREs) are 3′ UTR cis-regulatory elements that regulate the stability of mRNAs. Consensus ARE motifs have been determined, but little is known about how differences in 3′ UTR sequences that conform to these motifs affect their function. Here we use functional annotation of sequences from 3′ UTRs (fast-UTR), a massively parallel reporter assay (MPRA), to investigate the effects of 41,288 3′ UTR sequence fragments from 4,653 transcripts on gene expression and mRNA stability in Jurkat and Beas2B cells. Our analyses demonstrate that the length of an ARE and its registration (the first and last nucleotides of the repeating ARE motif) have significant effects on gene expression and stability. Based on this finding, we propose improved ARE classification and concomitant methods to categorize and predict the effect of AREs on gene expression and stability. Finally, to investigate the advantages of our general experimental design we examine other motifs including constitutive decay elements (CDEs), where we show that the length of the CDE stem-loop has a significant impact on steady-state expression and mRNA stability. We conclude that fast-UTR, in conjunction with our analytical approach, can produce improved yet simple sequence-based rules for predicting the activity of human 3′ UTRs.

scholarly journals Multinomial Convolutions for Joint Modeling of Sequence Motifs and Enhancer Activities

2020 ◽  
Author(s):  
Minjun Park ◽  
Salvi Singh ◽  
Francisco Jose Grisanti Canozo ◽  
Md. Abul Hassan Samee
Keyword(s):  
De Novo ◽  
Joint Modeling ◽  
Chromatin Accessibility ◽  
Massively Parallel ◽  
Sequence Motifs ◽  
Functional Variants ◽  
Transcriptional Regulatory ◽  
Reporter Assays ◽  
Transcriptional Regulatory Mechanisms ◽  
Model Sequence

AbstractMassively parallel reporter assays (MPRAs) have enabled the study of transcriptional regulatory mechanisms at an unprecedented scale and with high quantitative resolution. However, this realm lacks models that can discover sequence-specific signals de novo from the data and integrate them in a mechanistic way. We present MuSeAM (Multinomial CNNs for Sequence Activity Modeling), a convolutional neural network that overcomes this gap. MuSeAM utilizes multinomial convolutions that directly model sequence-specific motifs of protein-DNA binding. We demonstrate that MuSeAM fits MPRA data with high accuracy and generalizes over other tasks such as predicting chromatin accessibility and prioritizing potentially functional variants.

scholarly journals Deciphering the regulatory genome of Escherichia coli, one hundred promoters at a time

2020 ◽  
Author(s):  
William T. Ireland ◽  
Suzannah M. Beeler ◽  
Emanuel Flores-Bautista ◽  
Nathan M. Belliveau ◽  
Michael J. Sweredoski ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Conditions ◽  
Massively Parallel ◽  
Model Organisms ◽  
Reporter Assay ◽  
E Coli ◽  
Rosetta Stone ◽  
Regulatory Genome ◽  
Massively Parallel Reporter Assay ◽  
Medical Relevance

AbstractAdvances in DNA sequencing have revolutionized our ability to read genomes. However, even in the most well-studied of organisms, the bacterium Escherichia coli, for ≈ 65% of the promoters we remain completely ignorant of their regulation. Until we have cracked this regulatory Rosetta Stone, efforts to read and write genomes will remain haphazard. We introduce a new method (Reg-Seq) linking a massively-parallel reporter assay and mass spectrometry to produce a base pair resolution dissection of more than 100 promoters in E. coli in 12 different growth conditions. First, we show that our method recapitulates regulatory information from known sequences. Then, we examine the regulatory architectures for more than 80 promoters in the E. coli genome which previously had no known regulation. In many cases, we also identify which transcription factors mediate their regulation. The method introduced here clears a path for fully characterizing the regulatory genome of model organisms, with the potential of moving on to an array of other microbes of ecological and medical relevance.

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