scholarly journals Coupled single-cell CRISPR screening and epigenomic profiling reveals causal gene regulatory networks

2018 ◽  
Author(s):  
Adam J. Rubin ◽  
Kevin R. Parker ◽  
Ansuman T. Satpathy ◽  
Yanyan Qi ◽  
Beijing Wu ◽  
...  
Keyword(s):  
Gene Regulatory Networks ◽  
Regulatory Networks ◽  
Single Cells ◽  
Nucleosome Positioning ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Open Chromatin ◽  
General Strategy ◽  
List Type ◽  
Gene Regulatory

SummaryHere we present Perturb-ATAC, a method which combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells, based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in ∼4,300 single cells, encompassing more than 63 unique genotype-phenotype relationships. Perturb-ATAC in human B lymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning, and identified a hierarchical organization of TFs that govern B cell state, variation, and disease-associatedcis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules ofcis-elements that specify keratinocyte fate, orchestrated by the TFs JUNB, KLF4, ZNF750, CEBPA, and EHF. Combinatorial deletion of all pairs of these TFs uncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful and general strategy to dissect gene regulatory networks in development and disease.HighlightsA new method for simultaneous measurement of CRISPR perturbations and chromatin state in single cells.Perturb-ATAC reveals regulatory factors that controlcis-element accessibility,trans-factor occupancy, and nucleosome positioning.Perturb-ATAC reveals regulatory modules of coordinatedtrans-factor activity in B lymphoblasts.Keratinocyte differentiation is orchestrated by synergistic activities of co-binding TFs oncis-elements.

scholarly journals Distinct epicardial gene regulatory programmes drive development and regeneration of the zebrafish heart

2021 ◽  
Author(s):  
Michael Weinberger ◽  
Filipa C. Simoes ◽  
Tatjana Sauka-Spengler ◽  
Paul R. Riley
Keyword(s):  
Gene Regulatory Networks ◽  
Regulatory Networks ◽  
Expression Profiles ◽  
Regulatory Elements ◽  
Open Chromatin ◽  
Heart Regeneration ◽  
Mammalian Heart ◽  
Gene Regulatory ◽  
Zebrafish Heart

Unlike the adult mammalian heart, which has limited regenerative capacity, the zebrafish heart can fully regenerate following injury. Reactivation of cardiac developmental programmes is considered key to successfully regenerating the heart, yet the regulatory elements underlying the response triggered upon injury and during development remain elusive. Organ-wide activation of the epicardium is essential for zebrafish heart regeneration and is considered a potential regenerative source to target in the mammalian heart. Here we compared the transcriptome and epigenome of the developing and regenerating zebrafish epicardium by integrating gene expression profiles with open chromatin ATAC-seq data. By generating gene regulatory networks associated with epicardial development and regeneration, we inferred genetic programmes driving each of these processes, which were largely distinct. We identified wt1a, wt1b, and the AP-1 subunits junbb, fosab and fosb as central regulators of the developing network, whereas hif1ab, zbtb7a, tbx2b and nrf1 featured as putative central regulators of the regenerating epicardial network. By interrogating developmental gene regulatory networks that drive cell-specific transcriptional heterogeneity, we tested novel subpopulation-related epicardial enhancers in vivo. Taken together, our work revealed striking differences between the regulatory blueprint deployed during epicardial development and regeneration. These findings challenge the dogma that heart regeneration is essentially a reactivation of developmental programmes, and provide important insights into epicardial regulation that can assist in developing therapeutic approaches to enable tissue regeneration in the adult mammalian heart.

scholarly journals Integrative multi-omics analyses identify cell-type disease genes and regulatory networks across schizophrenia and Alzheimer’s disease

2020 ◽  
Author(s):  
Mufang Ying ◽  
Peter Rehani ◽  
Panagiotis Roussos ◽  
Daifeng Wang
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Gene Regulatory Networks ◽  
Regulatory Networks ◽  
Regulatory Elements ◽  
Disease Genes ◽  
Omics Data ◽  
Cell Type ◽  
Cellular Resolution ◽  
Gene Regulatory

AbstractStrong phenotype-genotype associations have been reported across brain diseases. However, understanding underlying gene regulatory mechanisms remains challenging, especially at the cellular level. To address this, we integrated the multi-omics data at the cellular resolution of the human brain: cell-type chromatin interactions, epigenomics and single cell transcriptomics, and predicted cell-type gene regulatory networks linking transcription factors, distal regulatory elements and target genes (e.g., excitatory and inhibitory neurons, microglia, oligodendrocyte). Using these cell-type networks and disease risk variants, we further identified the cell-type disease genes and regulatory networks for schizophrenia and Alzheimer’s disease. The celltype regulatory elements (e.g., enhancers) in the networks were also found to be potential pleiotropic regulatory loci for a variety of diseases. Further enrichment analyses including gene ontology and KEGG pathways revealed potential novel cross-disease and disease-specific molecular functions, advancing knowledge on the interplays among genetic, transcriptional and epigenetic risks at the cellular resolution between neurodegenerative and neuropsychiatric diseases. Finally, we summarized our computational analyses as a general-purpose pipeline for predicting gene regulatory networks via multi-omics data.

scholarly journals Mutations in Transcriptional Regulators Allow Selective Engineering of Signal Integration Logic

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Szabolcs Semsey
Keyword(s):  
Gene Regulatory Networks ◽  
Regulatory Networks ◽  
Regulatory Protein ◽  
Point Mutations ◽  
Building Blocks ◽  
Regulatory Elements ◽  
Regulatory Proteins ◽  
Single Amino Acid ◽  
Signal Integration ◽  
Gene Regulatory

ABSTRACT Bacterial cells monitor their environment by sensing a set of signals. Typically, these environmental signals affect promoter activities by altering the activity of transcription regulatory proteins. Promoters are often regulated by more than one regulatory protein, and in these cases the relevant signals are integrated by certain logic. In this work, we study how single amino acid substitutions in a regulatory protein (GalR) affect transcriptional regulation and signal integration logic at a set of engineered promoters. Our results suggest that point mutations in regulatory genes allow independent evolution of regulatory logic at different promoters. IMPORTANCE Gene regulatory networks are built from simple building blocks, such as promoters, transcription regulatory proteins, and their binding sites on DNA. Many promoters are regulated by more than one regulatory input. In these cases, the inputs are integrated and allow transcription only in certain combinations of input signals. Gene regulatory networks can be easily rewired, because the function of cis-regulatory elements and promoters can be altered by point mutations. In this work, we tested how point mutations in transcription regulatory proteins can affect signal integration logic. We found that such mutations allow context-dependent engineering of signal integration logic at promoters, further contributing to the plasticity of gene regulatory networks.

scholarly journals Synthetic gene-regulatory networks in the opportunistic human pathogenStreptococcus pneumoniae

2020 ◽  
Vol 117 (44) ◽  
pp. 27608-27619 ◽  
Author(s):  
Robin A. Sorg ◽  
Clement Gallay ◽  
Laurye Van Maele ◽  
Jean-Claude Sirard ◽  
Jan-Willem Veening
Keyword(s):  
Gene Expression ◽  
Virulence Factor ◽  
Gene Regulatory Networks ◽  
Regulatory Networks ◽  
Combinatorial Libraries ◽  
Regulatory Elements ◽  
Synthetic Gene ◽  
Expression Control ◽  
Gene Expression Control ◽  
Gene Regulatory

Streptococcus pneumoniaecan cause disease in various human tissues and organs, including the ear, the brain, the blood, and the lung, and thus in highly diverse and dynamic environments. It is challenging to study how pneumococci control virulence factor expression, because cues of natural environments and the presence of an immune system are difficult to simulate in vitro. Here, we apply synthetic biology methods to reverse-engineer gene expression control inS. pneumoniae. A selection platform is described that allows for straightforward identification of transcriptional regulatory elements out of combinatorial libraries. We present TetR- and LacI-regulated promoters that show expression ranges of four orders of magnitude. Based on these promoters, regulatory networks of higher complexity are assembled, such as logic AND gates and IMPLY gates. We demonstrate single-copy genome-integrated toggle switches that give rise to bimodal population distributions. The tools described here can be used to mimic complex expression patterns, such as the ones found for pneumococcal virulence factors. Indeed, we were able to rewire gene expression of the capsule operon, the main pneumococcal virulence factor, to be externally inducible (YES gate) or to act as an IMPLY gate (only expressed in absence of inducer). Importantly, we demonstrate that these synthetic gene-regulatory networks are functional in an influenza A virus superinfection murine model of pneumonia, paving the way for in vivo investigations of the importance of gene expression control on the pathogenicity ofS. pneumoniae.

scholarly journals Mapping gene regulatory networks of primary CD4+T cells using single-cell genomics and genome engineering

2019 ◽  
Author(s):  
Rachel E. Gate ◽  
Min Cheol Kim ◽  
Andrew Lu ◽  
David Lee ◽  
Eric Shifrut ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Gene Regulatory Networks ◽  
Regulatory Networks ◽  
Genome Engineering ◽  
Regulatory Elements ◽  
Mapping Gene ◽  
Gene Regulatory ◽  
Novel Interactions

AbstractGene regulatory programs controlling the activation and polarization of CD4+T cells are incompletely mapped and the interindividual variability in these programs remain unknown. We sequenced the transcriptomes of ~160k CD4+T cells from 9 donors following pooled CRISPR perturbation targeting 140 regulators. We identified 134 regulators that affect T cell functionalization, includingIRF2as a positive regulator of Th2polarization. Leveraging correlation patterns between cells, we mapped 194 pairs of interacting regulators, including known (e.g.BATFandJUN) and novel interactions (e.g.ETS1andSTAT6). Finally, we identified 80 natural genetic variants with effects on gene expression, 48 of which are modified by a perturbation. In CD4+T cells, CRISPR perturbations can influencein vitropolarization and modify the effects oftransandcisregulatory elements on gene expression.

scholarly journals Upregulation and cell specificity of C4 genes are derived from ancestral C3 gene regulatory networks

2020 ◽  
Author(s):  
Pallavi Singh ◽  
Sean R. Stevenson ◽  
Ivan Reyna-Llorens ◽  
Gregory Reeves ◽  
Tina B. Schreier ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Gene Regulatory Networks ◽  
Regulatory Networks ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
Transcript Accumulation ◽  
Specific Expression ◽  
Cell Specificity ◽  
Distinct Cell ◽  
Gene Regulatory

ABSTRACTThe efficient C4 pathway is based on strong up-regulation of genes found in C3 plants, but also compartmentation of their expression into distinct cell-types such as the mesophyll and bundle sheath. Transcription factors associated with these phenomena have not been identified. To address this, we undertook genome-wide analysis of transcript accumulation, chromatin accessibility and transcription factor binding in C4Gynandropsis gynandra. From these data, two models relating to the molecular evolution of C4 photosynthesis are proposed. First, increased expression of C4 genes is associated with increased binding by MYB-related transcription factors. Second, mesophyll specific expression is associated with binding of homeodomain transcription factors. Overall, we conclude that during evolution of the complex C4 trait, C4 cycle genes gain cis-elements that operate in the C3 leaf such that they become integrated into existing gene regulatory networks associated with cell specificity and photosynthesis.

scholarly journals Revealing the Key Regulators of Cell Identity in the Human Adult Pancreas

2020 ◽  
Author(s):  
Lotte Vanheer ◽  
Andrea Alex Schiavo ◽  
Matthias Van Haele ◽  
Tine Haesen ◽  
Adrian Janiszewski ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Gene Regulatory Networks ◽  
Regulatory Networks ◽  
Cell Types ◽  
List Type ◽  
Cell Identity ◽  
Pancreatic Cell ◽  
Human Adult ◽  
Gene Regulatory ◽  
Cellular Identity

SUMMARYCellular identity during development is under the control of transcription factors that form gene regulatory networks. However, the transcription factors and gene regulatory networks underlying cellular identity in the human adult pancreas remain largely unexplored. Here, we integrate multiple single-cell RNA sequencing datasets of the human adult pancreas, totaling 7393 cells, and comprehensively reconstruct gene regulatory networks. We show that a network of 142 transcription factors forms distinct regulatory modules that characterize pancreatic cell types. We present evidence that our approach identifies key regulators of cell identity in the human adult pancreas. We predict that HEYL and JUND are active in acinar and alpha cells, respectively, and show that these proteins are present in the human adult pancreas as well as in human induced pluripotent stem cell-derived pancreatic cells. The comprehensive gene regulatory network atlas can be explored interactively online. We anticipate our analysis to be the starting point for a more sophisticated dissection of how transcription factors regulate cell identity in the human adult pancreas. Furthermore, given that transcription factors are major regulators of embryo development and are often perturbed in diseases, a comprehensive understanding of how transcription factors work will be relevant in development and disease biology.HIGHLIGHTS-Reconstruction of gene regulatory networks for human adult pancreatic cell types-An interactive resource to explore and visualize gene expression and regulatory states-Predicting putative transcription factors driving pancreatic cell identity-HEYL and JUND as candidate regulators of acinar and alpha cell identity, respectively

scholarly journals Gene regulatory networks controlling vertebrate retinal regeneration

Science ◽  
2020 ◽  
Vol 370 (6519) ◽  
pp. eabb8598 ◽  
Author(s):  
Thanh Hoang ◽  
Jie Wang ◽  
Patrick Boyd ◽  
Fang Wang ◽  
Clayton Santiago ◽  
...  
Keyword(s):  
Gene Regulatory Networks ◽  
Gene Networks ◽  
Regulatory Networks ◽  
Chromatin Accessibility ◽  
Specific Gene ◽  
Muller Glia ◽  
Müller Glia ◽  
Retinal Regeneration ◽  
Adult Mice ◽  
Gene Regulatory

Injury induces retinal Müller glia of certain cold-blooded vertebrates, but not those of mammals, to regenerate neurons. To identify gene regulatory networks that reprogram Müller glia into progenitor cells, we profiled changes in gene expression and chromatin accessibility in Müller glia from zebrafish, chick, and mice in response to different stimuli. We identified evolutionarily conserved and species-specific gene networks controlling glial quiescence, reactivity, and neurogenesis. In zebrafish and chick, the transition from quiescence to reactivity is essential for retinal regeneration, whereas in mice, a dedicated network suppresses neurogenic competence and restores quiescence. Disruption of nuclear factor I transcription factors, which maintain and restore quiescence, induces Müller glia to proliferate and generate neurons in adult mice after injury. These findings may aid in designing therapies to restore retinal neurons lost to degenerative diseases.

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