STARR-seq of artifical enhancer seqeunces consisting of homotypic and heterotypic TF binding site repeats
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE229646 | GEO | 2024/06/30
REPOSITORIES: GEO
Similar Datasets
Project description:Massively parallel reporter assays (MPRAs) test the capacity of putative gene regulatory elements to drive transcription on a genome-wide scale. Most gene regulatory activity occurs within accessible chromatin, and recently described methods have combined assays that capture these regions—such as assay for transposase-accessible chromatin using sequencing (ATAC-seq)—with self-transcribing active regulatory region sequencing (STARR-seq) to selectively assay the regulatory potential of accessible DNA (ATAC-STARR-seq). Here, we report an integrated approach that quantifies activating and silencing regulatory activity, chromatin accessibility, and transcription factor (TF) occupancy with one assay using ATAC-STARR-seq. Our strategy, including important updates to the ATAC-STARR-seq assay and workflow, enabled high-resolution testing of ~50 million unique DNA fragments tiling ~101,000 accessible chromatin regions in human lymphoblastoid cells. We discovered that 30% of all accessible regions contain an activator, a silencer or both. Although few MPRA studies have explored silencing activity, we demonstrate silencers occur at similar frequencies to activators, and they represent a distinct functional group enriched for unique TF motifs and repressive histone modifications. We further show that Tn5 cut-site frequencies are retained in the ATAC-STARR plasmid library compared to standard ATAC-seq, enabling TF occupancy to be ascertained from ATAC-STARR data. With this approach, we found that activators and silencers cluster by distinct TF footprint combinations and these groups of activity represent different gene regulatory networks of immune cell function. Altogether, these data highlight the multi-layered capabilities of ATAC-STARR-seq to comprehensively investigate the regulatory landscape of the human genome all from a single DNA fragment source.
2022-07-06 | GSE181317 | GEO
Project description:Genomic enhancers are important regulators of gene expression, but their identification is a challenge and methods depend on indirect measures of activity. We developed a method termed STARR-seq to directly and quantitatively assess enhancer activity for millions of candidates from arbitrary sources of DNA, enabling screens across entire genomes. When applied to the Drosophila genome, STARR-seq identifies thousands of cell type-specific enhancers across a broad continuum of strengths, linking differential gene expression to differences in enhancer activity and creating a genome-wide quantitative enhancer map. This map reveals the highly complex regulation of transcription, with several independent enhancers for both developmental regulators and ubiquitously expressed genes. STARR-seq can be used to identify and quantitate enhancer activity in other eukaryotes, including human. STARR-seq was performed in S2 and OSC cells with paired-end sequencing in two replicates and respective inputs. DHS-seq was done with single-end sequencing in two replicates for S2 and OSC cells. RNA-seq was performed with a strand-specific protocol using single-end sequencing in two replicates within S2 and OSC cells. STARR-seq was also performed in HeLa cells with single-end sequencing with a respective input.
2013-01-17 | E-GEOD-40739 | biostudies-arrayexpress
Project description:Massively parallel reporter assays (MPRA) are widely used to discover functional enhancers but have largely been limited to transfected cell models. Here, we combine hydrodynamic injection with a modified STARR-seq-based MPRA to determine condition-specific enhancer activity in mouse liver at scale, and we examine how different promoters affect STARR-seq reporter activity. Strong liver enhancer activity was observed with STARR-seq libraries containing an Albumin minimal promoter but not when using a Super Core promoter or an origin of replication (ORI) promoter. This work is part of a larger study where we prepare a global STARR-seq library, comprised of ~50,000 genomic sequences released by DNase-I digestion of mouse liver nuclei, and where we identify condition-specific enhancers with strong correlations between liver enhancer activity and the chromatin state of the corresponding endogenous genomic regions.
2024-06-17 | GSE267075 | GEO
Project description:We combined Self-Transcribing Active Regulatory Region Sequencing (STARR-seq) with an enrichment step using chromatin immunoprecipitation in a massively parallel reporter assay. We applied this assay, termed ChIP-STARR-seq, to normal (primed) and naive human embryonic stem cells, building up a comprehensive catalogue of functional enhancers. This database record describes the STARR-RNA-seq component.
2017-07-05 | GSE99630 | GEO
Project description:Enhancers are important regulators of gene expression, but their identification is a challenge in plants. STARR-seq is a method measuring directly the enhancer activity of millions fragments in parallel, which had been successfully used to identify enhancers in Drosophila and human genomes. Here we present a global map of rice enhancers whose activities are quantitatively determined by STARR-seq.We also predicted intergenic enhancers based on DNase I hypersensitivity as described in a previously published work. Predicted enhancers overlap poorly with STARR-seq enhancers, only about 400 sites accounting for 3-4% of total enhancers identified by these two different methods. In summary, our results of STARR-seq reveal that enhancers in a plant genome differ from animal enhancers in several aspects and provide a regulatory element resource for further functional and mechanistic studies in different contexts
2018-11-08 | GSE121231 | GEO
Project description:Genomic enhancers are important regulators of gene expression, but their identification is a challenge and methods depend on indirect measures of activity. We developed a method termed STARR-seq to directly and quantitatively assess enhancer activity for millions of candidates from arbitrary sources of DNA, enabling screens across entire genomes. When applied to the Drosophila genome, STARR-seq identifies thousands of cell type-specific enhancers across a broad continuum of strengths, linking differential gene expression to differences in enhancer activity and creating a genome-wide quantitative enhancer map. This map reveals the highly complex regulation of transcription, with several independent enhancers for both developmental regulators and ubiquitously expressed genes. STARR-seq can be used to identify and quantitate enhancer activity in other eukaryotes, including human.
2013-01-17 | GSE40739 | GEO
Project description:Tissue engineering can potentially recreate in vivo cellular microenvironments in vitro for an array of applications such as biological inquiry and drug discovery. However, the majority of current in vitro systems still neglect many biological, chemical, and mechanical cues that are known to impact cellular functions such as proliferation, migration, and differentiation. To address this gap, we have developed a novel microfluidic device that precisely controls the spatial and temporal interactions between adjacent three-dimensional cellular environments. The device consists of four interconnected microtissue compartments (~0.1 mm(3)) arranged in a square. The top and bottom pairs of compartments can be sequentially loaded with discrete cellularized hydrogels creating the opportunity to investigate homotypic (left to right or x-direction) and heterotypic (top to bottom or y-direction) cell-cell communication. A controlled hydrostatic pressure difference across the tissue compartments in both x and y direction induces interstitial flow and modulates communication via soluble factors. To validate the biological significance of this novel platform, we examined the role of stromal cells in the process of vasculogenesis. Our device confirms previous observations that soluble mediators derived from normal human lung fibroblasts (NHLFs) are necessary to form a vascular network derived from endothelial colony forming cell-derived endothelial cells (ECFC-ECs). We conclude that this platform could be used to study important physiological and pathological processes that rely on homotypic and heterotypic cell-cell communication.
| S-EPMC4855298 | biostudies-literature
Project description:The mammalian pseudokinase SgK223, and its structurally related homologue SgK269, are oncogenic scaffolds that nucleate the assembly of specific signalling complexes and regulate tyrosine kinase signalling. Both SgK223 and SgK269 form homo- and hetero-oligomers, a mechanism that underpins a diversity of signalling outputs. However, mechanistic insights into SgK223 and SgK269 homo- and heterotypic association are lacking. Here we present the crystal structure of SgK223 pseudokinase domain and its adjacent N- and C-terminal helices. The structure reveals how the N- and C-regulatory helices engage in a novel fold to mediate the assembly of a high-affinity dimer. In addition, we identified regulatory interfaces on the pseudokinase domain required for the self-assembly of large open-ended oligomers. This study highlights the diversity in how the kinase fold mediates non-catalytic functions and provides mechanistic insights into how the assembly of these two oncogenic scaffolds is achieved in order to regulate signalling output.
| S-EPMC5660093 | biostudies-literature
Project description:Proteasome-mediated degradation occurs with proteins principally modified with lysine-48 polyubiquitin chains. Whether the proteasome also can bind atypical ubiquitin chains, including those linked by lysine-11, has not been well established. This is critically important, as lysine-11 polyubiquitination has been implicated in both proteasome-mediated degradation and non-degradative outcomes. Here we demonstrate that pure homotypic lysine-11-linked chains do not bind strongly to the mammalian proteasome. By contrast, heterotypic polyubiquitin chains, containing lysine-11 and lysine-48 linkages, not only bind to the proteasome but also stimulate the proteasomal degradation of the cell-cycle regulator cyclin B1. Thus, while heterotypic lysine-11-linked chains facilitate proteasomal degradation, homotypic lysine-11 linkages adopt conformations that prevent association with the proteasome. Our data demonstrate the capacity of the proteasome to bind ubiquitin chains of distinct topology, with implications for the recognition and diverse biological functions of mixed ubiquitin chains.
| S-EPMC4533228 | biostudies-literature
Project description:We performed a STARR-seq based, massively parallel reporter assay on reference genome and mutation-containing oligonucleotide sequences from regions associated with a large number of transcription factor ChIP-seq peaks or DHS-footprinted motifs.
2019-12-25 | GSE142566 | GEO