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 Massively parallel discovery of human-specific substitutions that alter enhancer activity

2020 ◽  
Vol 118 (2) ◽  
pp. e2007049118
Author(s):  
Severin Uebbing ◽  
Jake Gockley ◽  
Steven K. Reilly ◽  
Acadia A. Kocher ◽  
Evan Geller ◽  
...  
Keyword(s):  
Human Evolution ◽  
Regulatory Elements ◽  
Massively Parallel ◽  
Enhancer Activity ◽  
Genetic Changes ◽  
Gene Regulatory Elements ◽  
Enhancer Function ◽  
Human Accelerated Regions ◽  
The Impact ◽  
Human Specific

Genetic changes that altered the function of gene regulatory elements have been implicated in the evolution of human traits such as the expansion of the cerebral cortex. However, identifying the particular changes that modified regulatory activity during human evolution remain challenging. Here we used massively parallel enhancer assays in neural stem cells to quantify the functional impact of >32,000 human-specific substitutions in >4,300 human accelerated regions (HARs) and human gain enhancers (HGEs), which include enhancers with novel activities in humans. We found that >30% of active HARs and HGEs exhibited differential activity between human and chimpanzee. We isolated the effects of human-specific substitutions from background genetic variation to identify the effects of genetic changes most relevant to human evolution. We found that substitutions interacted in both additive and nonadditive ways to modify enhancer function. Substitutions within HARs, which are highly constrained compared to HGEs, showed smaller effects on enhancer activity, suggesting that the impact of human-specific substitutions is buffered in enhancers with constrained ancestral functions. Our findings yield insight into how human-specific genetic changes altered enhancer function and provide a rich set of candidates for studies of regulatory evolution in humans.

scholarly journals Massively parallel discovery of human-specific substitutions that alter neurodevelopmental enhancer activity

2019 ◽  
Author(s):  
Severin Uebbing ◽  
Jake Gockley ◽  
Steven K. Reilly ◽  
Acadia A. Kocher ◽  
Evan Geller ◽  
...  
Keyword(s):  
Binding Sites ◽  
Experimental Studies ◽  
Regulatory Elements ◽  
Massively Parallel ◽  
Enhancer Activity ◽  
Genetic Changes ◽  
Human Neural Stem Cells ◽  
Human Accelerated Regions ◽  
The Impact ◽  
Human Specific

AbstractGenetic changes that altered the function of gene regulatory elements have been implicated in the evolution of the human brain. However, identifying the particular changes that modified regulatory activity during neurodevelopment remains challenging. Here we used massively parallel enhancer assays in human neural stem cells to measure the impact of 32,776 human-specific substitutions on enhancer activity in 1,363 Human Accelerated Regions (HARs) and 3,027 Human Gain Enhancers (HGEs), which include enhancers with novel activities in humans. We found that 31.9% of active HARs and 36.4% of active HGEs exhibited differential activity between human and chimpanzee. This enabled us to isolate the effects of 401 human-specific substitutions from other types of genetic variation in HARs and HGEs. Substitutions acted in both an additive and non-additive manner to alter enhancer activity. Human-specific substitutions altered predicted binding sites for a specific set of human transcription factors (TFs) that were a subset of TF binding sites associated with enhancer activity in our assay. Substitutions within HARs, which are overall highly constrained compared to HGEs, showed smaller effects on enhancer activity, suggesting that the impact of human-specific substitutions may be buffered in enhancers with constrained ancestral functions. Our findings yield insight into the mechanisms by which human-specific genetic changes impact enhancer function and provide a rich set of candidates for experimental studies of regulatory evolution in humans.

Evolutionary Changes in Transcriptional Regulation: Insights into Human Behavior and Neurological Conditions

2018 ◽  
Vol 41 (1) ◽  
pp. 185-206 ◽  
Author(s):  
Ryan N. Doan ◽  
Taehwan Shin ◽  
Christopher A. Walsh
Keyword(s):  
Population Data ◽  
Functional Information ◽  
Human Lineage ◽  
Medical Genetic ◽  
Noncoding Sequences ◽  
Noncoding Regions ◽  
Evolutionary Changes ◽  
Neurological Conditions ◽  
Human Accelerated Regions ◽  
Human Specific

Understanding the biological basis for human-specific cognitive traits presents both immense challenges and unique opportunities. Although the question of what makes us human has been investigated with several different methods, the rise of comparative genomics, epigenomics, and medical genetics has provided tools to help narrow down and functionally assess the regions of the genome that seem evolutionarily relevant along the human lineage. In this review, we focus on how medical genetic cases have provided compelling functional evidence for genes and loci that appear to have interesting evolutionary signatures in humans. Furthermore, we examine a special class of noncoding regions, human accelerated regions (HARs), that have been suggested to show human-lineage-specific divergence, and how the use of clinical and population data has started to provide functional information to examine these regions. Finally, we outline methods that provide new insights into functional noncoding sequences in evolution.

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 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 Developmental loci harbor clusters of accelerated regions that evolved independently in ape lineages

2017 ◽  
Author(s):  
Dennis Kostka ◽  
Alisha K. Holloway ◽  
Katherine S. Pollard
Keyword(s):  
Statistical Tests ◽  
Regulatory Elements ◽  
Altered Expression ◽  
Evolutionary Forces ◽  
Shared Set ◽  
Accelerated Evolution ◽  
Core Set ◽  
Regulatory Dna ◽  
Human Accelerated Regions ◽  
Human Specific

AbstractSome of the fastest evolving regions of the human genome are conserved non-coding elements with many human-specific DNA substitutions. These Human Accelerated Regions (HARs) are enriched nearby regulatory genes, and several HARs function as developmental enhancers. To investigate if this evolutionary signature is unique to humans, we quantified evidence of accelerated substitutions in conserved genomic elements across multiple lineages and applied this approach simultaneously to the genomes of five apes: human, chimpanzee, gorilla, orangutan, and gibbon. We find roughly similar numbers and genomic distributions of lineage-specific accelerated regions (linARs) in all five apes. In particular, apes share an enrichment of linARs in regulatory DNA nearby genes involved in development, especially transcription factors and other regulators. Many developmental loci harbor clusters of nonoverlapping linARs from multiple apes, suggesting that accelerated evolution in each species affected distinct regulatory elements that control a shared set of developmental pathways. Our statistical tests distinguish between GC-biased and unbiased accelerated substitution rates, allowing us to quantify the roles of different evolutionary forces in creating linARs. We find evidence of GC-biased gene conversion in each ape, but unbiased acceleration consistent with positive selection or loss of constraint is more common in all five lineages. It therefore appears that similar evolutionary processes created independent accelerated regions in the genomes of different apes, and that these lineage-specific changes to conserved non-coding sequences may have differentially altered expression of a core set of developmental genes across ape evolution.

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 Human Accelerated Region participates in early human forebrain patterning and expansion

2019 ◽  
Author(s):  
Sandra Acosta ◽  
Jaydeep Sidhaye ◽  
Luciano Fiore ◽  
Isabel Rollan ◽  
Giovanni Iacono ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Developmental Stages ◽  
Regulatory Elements ◽  
Mammalian Brain ◽  
Enhancer Activity ◽  
Evolutionary Changes ◽  
Active Enhancer ◽  
Phylotypic Stage ◽  
Early Developmental ◽  
Human Specific

AbstractThe expansion of the mammalian brain is associated with specific developmental processes; however, not much is known about how evolutionary changes participated in the acquisition of human brain traits during early developmental stages. Here we investigated whether enhancers active during the phylotypic stage show human-specific genomic divergence which could contribute to the evolutionary expansion of the forebrain. Notably, we identified an active enhancer containing a human accelerated region (HAR) located in the Chromosome 14q12, a region enriched with neurodevelopmental genes, such as Foxg1, Nkx2.1 and Nova1. Reporter analysis revealed that the human variant is active in the forebrain in transgenic mice and that it has stronger enhancer activity than the mouse or chimpanzee versions. Humanization of the mouse enhancer variant in transgenic mice and in mouse organoids resulted in an expansion of Foxg1 expressing domains in the forebrain early neural progenitors with a bias towards dorsal identities. Overall, our results suggest that human-specific mutations in critical regulatory elements controlling early brain development impact the expansion and patterning of the forebrain.

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.

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