Elsevier eBooks, Год журнала: 2024, Номер unknown
Опубликована: Янв. 1, 2024
Язык: Английский
Scientific Reports, Год журнала: 2024, Номер 14(1)
Опубликована: Фев. 16, 2024
Regulation of gene expression through enhancers is one the major processes shaping structure and function human brain during development. High-throughput assays have predicted thousands involved in neurodevelopment, confirming their activity orthogonal functional crucial. Here, we utilized Massively Parallel Reporter Assays (MPRAs) stem cells forebrain organoids to evaluate ~ 7000 gene-linked previously identified fetal tissues organoids. We used a Gaussian mixture model contribution background noise measured signal confirm 35% tested enhancers, with most showing temporal-specific activity, suggesting evolving role neurodevelopment. The temporal specificity was further supported by correlation expression. Our findings provide valuable regulatory resource scientific community.
Язык: Английский
Процитировано
9
Science, Год журнала: 2025, Номер 387(6733)
Опубликована: Янв. 30, 2025
Studying the functional consequences of structural variants (SVs) in mammalian genomes is challenging because (i) SVs arise much less commonly than single-nucleotide or small indels and (ii) methods to generate, map, characterize model systems are underdeveloped. To address these challenges, we developed Genome-Shuffle-seq, a method that enables multiplex generation mapping thousands (deletions, inversions, translocations, extrachromosomal circles) throughout genomes. We also demonstrate co-capture SV identity with single-cell transcriptomes, facilitating measurement impact on gene expression. anticipate Genome-Shuffle-seq will be broadly useful for systematic exploration expression, chromatin landscape, three-dimensional nuclear architecture, while initiating path toward minimal genome.
Язык: Английский
Процитировано
1
Genome biology, Год журнала: 2023, Номер 24(1)
Опубликована: Май 12, 2023
Enhancers are genomic DNA elements controlling spatiotemporal gene expression. Their flexible organization and functional redundancies make deciphering their sequence-function relationships challenging. This article provides an overview of the current understanding enhancer evolution, with emphasis on factors that influence these relationships. Technological advancements, particularly in machine learning synthetic biology, discussed light how they provide new ways to understand this complexity. Exciting opportunities lie ahead as we continue unravel intricacies function.
Язык: Английский
Процитировано
18
bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown
Опубликована: Фев. 15, 2023
Abstract Nucleotide changes in gene regulatory elements are important determinants of neuronal development and disease. Using massively parallel reporter assays primary human cells from mid-gestation cortex cerebral organoids, we interrogated the cis -regulatory activity 102,767 sequences, including differentially accessible cell-type specific regions developing single-nucleotide variants associated with psychiatric disorders. In cells, identified 46,802 active enhancer sequences 164 disorder-associated that significantly alter activity. Activity was comparable organoids suggesting provide an adequate model for cortex. deep learning, decoded sequence basis upstream regulators This work establishes a comprehensive catalog functional development. One Sentence Summary We identify enhancers disorder effects organoids.
Язык: Английский
Процитировано
16
bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown
Опубликована: Янв. 23, 2024
Abstract The functional consequences of structural variants (SVs) in mammalian genomes are challenging to study. This is due several factors, including: 1) their numerical paucity relative other forms standing genetic variation such as single nucleotide (SNVs) and short insertions or deletions (indels); 2) the fact that a SV can involve potentially impact function more than one gene and/or cis regulatory element; 3) immaturity methods generate map SVs, either randomly targeted fashion, vitro vivo model systems. Towards addressing these challenges, we developed Genome-Shuffle-seq , straightforward method enables multiplex generation mapping major SVs (deletions, inversions, translocations) throughout genome. based on integration “shuffle cassettes’’ genome, wherein each shuffle cassette contains components facilitate its site-specific recombination (SSR) with integrated cassettes (via Cre-loxP), specific genomic location T7-mediated transcription IVT), identification single-cell RNA-seq (scRNA-seq) data situ IST). In this proof-of-concept, apply induce thousands mouse embryonic stem cells (mESCs) experiment. Induced rapidly depleted from cellular population over time, possibly Cre-mediated toxicity negative selection rearrangements themselves. Leveraging T7 IST barcodes whose positions already mapped, further demonstrate efficiently genotype which present association many cell transcriptomes scRNA-seq data. Finally, preliminary evidence suggests our may be powerful means generating extrachromosomal circular DNAs (ecDNAs). Looking forward, anticipate broadly useful for systematic exploration expression, chromatin landscape, 3D nuclear architecture. We potential uses modeling ecDNAs, well paving path minimal
Язык: Английский
Процитировано
7
bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown
Опубликована: Июнь 14, 2024
Summary An important and largely unsolved problem in synthetic biology is how to target gene expression specific cell types. Here, we apply iterative deep learning design enhancers with strong differential activity between two human lines. We initially train models on published datasets of enhancer chromatin accessibility use them guide the that maximize predicted specificity. experimentally validate these sequences, measurements re-optimize predictor, a second generation improved Our methods embed relevant transcription factor binding site (TFBS) motifs higher frequencies than comparable endogenous while using more selective motif vocabulary, show correlated at single level. Finally, characterize causal features top via perturbation experiments as short 50bp can maintain
Язык: Английский
Процитировано
6
BioEssays, Год журнала: 2023, Номер 45(10)
Опубликована: Май 31, 2023
Tight control of the transcription process is essential for correct spatial and temporal gene expression pattern during development in homeostasis. Enhancers are at core transcriptional activation. The original definition an enhancer straightforward: a DNA sequence that activates independent orientation direction. Dissection numerous loci has shown many enhancer-like elements might not conform to definition, suggesting enhancers use multiple different mechanisms contribute Here, we review methodologies identify discuss pitfalls consequences our understanding regulation.
Язык: Английский
Процитировано
11
bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown
Опубликована: Янв. 16, 2025
Abstract Cis -acting regulatory enhancer elements are valuable tools for gaining cell type-specific genetic access. Leveraging large chromatin accessibility atlases, putative sequences can be identified and deployed in adeno-associated virus (AAV) delivery platforms. However, a significant bottleneck AAV discovery is charting their detailed expression patterns vivo , process that currently requires gold-standard one-by-one testing. Here we present barcoded multiplex strategy screening AAVs at type resolution using single RNA sequencing taxonomy mapping. We executed proof-of-concept study small pool of validated expressing variety neuronal non-neuronal types across the mouse brain. Unexpectedly, encountered substantial technical biological noise including chimeric packaging products, necessitating development novel techniques to accurately deconvolve patterns. These results underscore need improved methods mitigate highlight complexity biology .
Язык: Английский
Процитировано
0
eLife, Год журнала: 2023, Номер 12
Опубликована: Июль 27, 2023
Despite ground-breaking genetic studies that have identified thousands of risk variants for developmental diseases, how these lead to molecular and cellular phenotypes remains a gap in knowledge. Many are non-coding occur at enhancers, which orchestrate key regulatory programs during development. The prevailing paradigm is alter the activity impacting gene expression programs, ultimately contributing disease risk. A obstacle progress systematic functional characterization scale, especially since enhancer highly specific cell type stage. Here, we review foundational enhancers current genomic approaches functionally characterize their scale. In coming decade, anticipate perturbation link mechanisms, changes state, phenotypes.
Язык: Английский
Процитировано
7
bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown
Опубликована: Июнь 22, 2024
Genome-wide CRISPR-Cas9 screens have untangled regulatory networks and revealed the genetic underpinnings of diverse biological processes. Their success relies on experimental designs that interrogate specific molecular phenotypes distinguish key regulators from background effects. Here, we realize these goals with a generalizable platform for CRISPR interference barcoded expression reporter sequencing (CiBER-seq) dramatically improves sensitivity scope genome-wide screens. We systematically address technical factors distort phenotypic measurements by normalizing reporters against closely-matched control promoters, integrated together into genome at single copy. To test our ability to capture post-transcriptional post-translational regulation through sequencing, screened genes affected nonsense-mediated mRNA decay Doa10-mediated cytosolic protein decay. Our optimized CiBER-seq accurately known components well-studied RNA quality pathways minimal background. These results demonstrate precision versatility dissecting controlling cellular behaviors.
Язык: Английский
Процитировано
2