scholarly journals Analysis of long and short enhancers in melanoma cell states

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
David Mauduit ◽  
Ibrahim Ihsan Taskiran ◽  
Liesbeth Minnoye ◽  
Maxime de Waegeneer ◽  
Valerie Christiaens ◽  
...  
Keyword(s):  
Melanoma Cell ◽  
Massively Parallel ◽  
Cell Type ◽  
Cell Type Specificity ◽  
Enhancer Activity ◽  
Reporter Assays ◽  
Enhancer Function ◽  
The Relationship ◽  
Deep Learning Model ◽  
Shed Light

Understanding how enhancers drive cell type specificity and efficiently identifying them is essential for the development of innovative therapeutic strategies. In melanoma, the melanocytic (MEL) and the mesenchymal-like (MES) states present themselves with different responses to therapy, making the identification of specific enhancers highly relevant. Using massively parallel reporter assays (MPRA) in a panel of patient-derived melanoma lines (MM lines), we set to identify and decipher melanoma enhancers by first focusing on regions with state specific H3K27 acetylation close to differentially expressed genes. An in-depth evaluation of those regions was then pursued by investigating the activity of overlapping ATAC-seq peaks along with a full tiling of the acetylated regions with 190 bp sequences. Activity was observed in more than 60% of the selected regions and we were able to precisely locate the active enhancers within ATAC-seq peaks. Comparison of sequence content with activity, using the deep learning model DeepMEL2, revealed that AP-1 alone is responsible for the MES enhancer activity. In contrast, SOX10 and MITF both influence MEL enhancer function with SOX10 being required to achieve high levels of activity. Overall, our MPRAs shed light on the relationship between long and short sequences in terms of their sequence content, enhancer activity, and specificity across melanoma cell states.

scholarly journals Analysis of long and short enhancers in melanoma cell states

2021 ◽  
Author(s):  
David Mauduit ◽  
Liesbeth Minnoye ◽  
Ibrahim Ihsan Taskiran ◽  
Maxime De Waegeneer ◽  
Valerie Christiaens ◽  
...  
Keyword(s):  
Melanoma Cell ◽  
Active Regions ◽  
Massively Parallel ◽  
Cell Type ◽  
Cell Type Specificity ◽  
Enhancer Activity ◽  
Massively Parallel Reporter Assay ◽  
The Relationship ◽  
Deep Learning Model ◽  
Shed Light

Understanding how enhancers drive cell type specificity and efficiently identifying them is essential for the development of innovative therapeutic strategies. In melanoma, the melanocytic (MEL) and the mesenchymal-like (MES) states present themselves with different responses to therapy, making the identification of specific enhancers highly relevant. Using massively parallel reporter assay (MPRA) in a panel of patient-derived melanoma lines (MM lines), we set to identify and decipher melanoma enhancers by first focusing on regions with state specific H3K27 acetylation close to differentially expressed genes. A more in-depth evaluation of those regions was then pursued by investigating the activity of ATAC-seq peaks found therein along with a full tiling of the acetylated regions with 190 bp sequences. Activity was observed in more than 60% of the selected regions and we were able to precisely locate the active regions within ATAC-seq peaks. Comparison of sequence content with activity, using the deep learning model DeepMEL2, revealed that AP-1 alone is responsible for the MES enhancer activity, while SOX and MITF both influence MEL enhancer activity with SOX being required to achieve high levels of activity. Overall, our MPRA assays shed light on the relationship between long and short sequences in terms of their sequence content, enhancer activity, and specificity as reporters across melanoma cell states.

scholarly journals DeepSTARR predicts enhancer activity from DNA sequence and enables the de novo design of enhancers

2021 ◽  
Author(s):  
Bernardo P de Almeida ◽  
Franziska Reiter ◽  
Michaela Pagani ◽  
Alexander Stark
Keyword(s):  
Dna Sequence ◽  
Binding Sites ◽  
De Novo ◽  
De Novo Design ◽  
Flanking Sequence ◽  
Enhancer Activity ◽  
Drosophila Melanogaster S2 Cells ◽  
Enhancer Sequences ◽  
The Relationship ◽  
Deep Learning Model

Enhancer sequences control gene expression and comprise binding sites (motifs) for different transcription factors (TFs). Despite extensive genetic and computational studies, the relationship between DNA sequence and regulatory activity is poorly understood and enhancer de novo design is considered impossible. Here we built a deep learning model, DeepSTARR, to quantitatively predict the activities of thousands of developmental and housekeeping enhancers directly from DNA sequence in Drosophila melanogaster S2 cells. The model learned relevant TF motifs and higher-order syntax rules, including functionally non-equivalent instances of the same TF motif that are determined by motif-flanking sequence and inter-motif distances. We validated these rules experimentally and demonstrated their conservation in human by testing more than 40,000 wildtype and mutant Drosophila and human enhancers. Finally, we designed and functionally validated synthetic enhancers with desired activities de novo.

scholarly journals Systematic dissection of genomic features determining transcription factor binding and enhancer function

2017 ◽  
Vol 114 (7) ◽  
pp. E1291-E1300 ◽  
Author(s):  
Sharon R. Grossman ◽  
Xiaolan Zhang ◽  
Li Wang ◽  
Jesse Engreitz ◽  
Alexandre Melnikov ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Binding Assays ◽  
Genomic Locus ◽  
Enhancer Activity ◽  
Genomic Features ◽  
Motif Site ◽  
Reporter Assays ◽  
Enhancer Function

Enhancers regulate gene expression through the binding of sequence-specific transcription factors (TFs) to cognate motifs. Various features influence TF binding and enhancer function—including the chromatin state of the genomic locus, the affinities of the binding site, the activity of the bound TFs, and interactions among TFs. However, the precise nature and relative contributions of these features remain unclear. Here, we used massively parallel reporter assays (MPRAs) involving 32,115 natural and synthetic enhancers, together with high-throughput in vivo binding assays, to systematically dissect the contribution of each of these features to the binding and activity of genomic regulatory elements that contain motifs for PPARγ, a TF that serves as a key regulator of adipogenesis. We show that distinct sets of features govern PPARγ binding vs. enhancer activity. PPARγ binding is largely governed by the affinity of the specific motif site and higher-order features of the larger genomic locus, such as chromatin accessibility. In contrast, the enhancer activity of PPARγ binding sites depends on varying contributions from dozens of TFs in the immediate vicinity, including interactions between combinations of these TFs. Different pairs of motifs follow different interaction rules, including subadditive, additive, and superadditive interactions among specific classes of TFs, with both spatially constrained and flexible grammars. Our results provide a paradigm for the systematic characterization of the genomic features underlying regulatory elements, applicable to the design of synthetic regulatory elements or the interpretation of human genetic variation.

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 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 Recent advances in high-throughput approaches to dissect enhancer function

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 939 ◽  
Author(s):  
David Santiago-Algarra ◽  
Lan T.M. Dao ◽  
Lydie Pradel ◽  
Alexandre España ◽  
Salvatore Spicuglia
Keyword(s):  
High Throughput ◽  
Regulatory Elements ◽  
Genetic Alterations ◽  
Regulatory Sequences ◽  
Transcription Start ◽  
Enhancer Activity ◽  
Recent Advances ◽  
Reporter Assays ◽  
Enhancer Function ◽  
Higher Eukaryotes

The regulation of gene transcription in higher eukaryotes is accomplished through the involvement of transcription start site (TSS)-proximal (promoters) and -distal (enhancers) regulatory elements. It is now well acknowledged that enhancer elements play an essential role during development and cell differentiation, while genetic alterations in these elements are a major cause of human disease. Many strategies have been developed to identify and characterize enhancers. Here, we discuss recent advances in high-throughput approaches to assess enhancer activity, from the well-established massively parallel reporter assays to the recent clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based technologies. We highlight how these approaches contribute toward a better understanding of enhancer function, eventually leading to the discovery of new types of regulatory sequences, and how the alteration of enhancers can affect transcriptional regulation.

scholarly journals PESCA: A scalable platform for the development of cell-type-specific viral drivers

2019 ◽  
Author(s):  
Sinisa Hrvatin ◽  
Christopher P. Tzeng ◽  
M. Aurel Nagy ◽  
Hume Stroud ◽  
Charalampia Koutsioumpa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heterologous Gene Expression ◽  
Single Cells ◽  
Cell Types ◽  
Regulatory Elements ◽  
Functional Evaluation ◽  
Cell Type ◽  
Cell Type Specificity ◽  
Enhancer Activity ◽  
Cell Type Specific

AbstractEnhancers are the primary DNA regulatory elements that confer cell type specificity of gene expression. Recent studies characterizing individual enhancers have revealed their potential to direct heterologous gene expression in a highly cell-type-specific manner. However, it has not yet been possible to systematically identify and test the function of enhancers for each of the many cell types in an organism. We have developed PESCA, a scalable and generalizable method that leverages ATAC- and single-cell RNA-sequencing protocols, to characterize cell-type-specific enhancers that should enable genetic access and perturbation of gene function across mammalian cell types. Focusing on the highly heterogeneous mammalian cerebral cortex, we apply PESCA to find enhancers and generate viral reagents capable of accessing and manipulating a subset of somatostatin-expressing cortical interneurons with high specificity. This study demonstrates the utility of this platform for developing new cell-type-specific viral reagents, with significant implications for both basic and translational research.One sentence summaryHighly paralleled functional evaluation of enhancer activity in single cells generates new cell-type-specific tools with broad medical and scientific applications.

scholarly journals Identification of blood autosomal cis-expression quantitative trait methylation (cis-eQTMs) in children

2020 ◽  
Author(s):  
Carlos Ruiz-Arenas ◽  
Carles Hernandez-Ferrer ◽  
Marta Vives-Usano ◽  
Sergi Marí ◽  
Inés Quintela ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Quantitative Trait ◽  
Association Studies ◽  
Cell Type ◽  
Cell Type Specificity ◽  
Biological Interpretation ◽  
Cell Type Specific ◽  
Using Data ◽  
The Relationship

AbstractBackgroundThe identification of expression quantitative trait methylation (eQTMs), defined as correlations between gene expression and DNA methylation levels, might help the biological interpretation of epigenome-wide association studies (EWAS). We aimed to identify autosomal cis-eQTMs in child blood, using data from 832 children of the Human Early Life Exposome (HELIX) project.MethodsBlood DNA methylation and gene expression were measured with the Illumina 450K and the Affymetrix HTA v2 arrays, respectively. The relationship between methylation levels and expression of nearby genes (transcription start site (TSS) within a window of 1 Mb) was assessed by fitting 13.6 M linear regressions adjusting for sex, age, and cohort.ResultsWe identified 63,831 autosomal cis-eQTMs, representing 35,228 unique CpGs and 11,071 unique transcript clusters (TCs, genes). 74.3% of these cis-eQTMs were located at <250 kb, 60.0% showed an inverse relationship and 23.9% had at least one genetic variant associated with the methylation and expression levels. They were enriched for active blood regulatory regions. Adjusting for cellular composition decreased the number of cis-eQTMs to 37.7%, suggesting that some of them were cell type-specific. The overlap of child blood cis-eQTMs with those described in adults was small, and child and adult shared cis-eQTMs tended to be proximal to the TSS, enriched for genetic variants and with lower cell type specificity. Only half of the cis-eQTMs could be captured through annotation to the closest gene.ConclusionsThis catalogue of blood autosomal cis-eQTMs in children can help the biological interpretation of EWAS findings, and is publicly available at https://helixomics.isglobal.org/.

scholarly journals Massively parallel reporter assays combined with cell-type specific eQTL informed multiple melanoma loci and identified a pleiotropic function of HIV-1 restriction gene,MX2, in melanoma promotion

2019 ◽  
Author(s):  
Jiyeon Choi ◽  
Tongwu Zhang ◽  
Andrew Vu ◽  
Julien Ablain ◽  
Matthew M Makowski ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Susceptibility Genes ◽  
Massively Parallel ◽  
Chromosome Band ◽  
Cell Type ◽  
Functional Variants ◽  
Reporter Assays ◽  
Cell Type Specific ◽  
Hiv 1 ◽  
Pleiotropic Function

AbstractGenome-wide association studies (GWAS) have identified ∼20 melanoma susceptibility loci. To identify susceptibility genes and variants simultaneously from multiple GWAS loci, we integrated massively-parallel reporter assays (MPRA) with cell type-specific epigenomic data as well as melanocyte-specific expression quantitative trait loci (eQTL) profiling. Starting from 16 melanoma loci, we selected 832 variants overlapping active regions of chromatin in cells of melanocytic lineage and identified 39 candidate functional variants displaying allelic transcriptional activity by MPRA. For four of these loci, we further identified four colocalizing melanocytecis-eQTL genes (CTSS,CASP8,MX2, andMAFF) matching the allelic activity of MPRA functional variants. Among these, we further characterized the locus encompassing the HIV-1 restriction gene,MX2, on chromosome band Chr21q22.3 and validated a functional variant, rs398206, among multiple high LD variants. rs398206 mediates allelic transcriptional activity via binding of the transcription factor, YY1. This allelic transcriptional regulation is consistent with a significantcis-eQTL ofMX2in primary human melanocytes, where the melanoma risk-associated A allele of rs398206 is correlated with higherMX2levels. Melanocyte-specific transgenic expression of humanMX2in a zebrafish model demonstrated accelerated melanoma formation in aBRAFV600Ebackground. Thus, using an efficient scalable approach to streamline GWAS follow-up functional studies, we identified multiple candidate melanoma susceptibility genes and variants, and uncovered a pleiotropic function ofMX2in melanoma susceptibility.

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.

Sign in / Sign up

Export Citation Format

Share Document