Cis-regulatory analysis of Onecut1 expression in fate-restricted retinal progenitor cells

BackgroundThe vertebrate retina consists of six major classes of neuronal cells. During development, these cells are generated from a pool of multipotent retinal progenitor cells (RPCs) that express the gene Vsx2. Fate-restricted RPCs have recently been identified, with limited mitotic potential and cell fate possibilities compared to multipotent RPCs. One population of fate-restricted RPCs, marked by activity of the regulatory element ThrbCRM1, gives rise to both cone photoreceptors and horizontal cells. These cells do not express Vsx2, but co-express the transcription factors (TFs) Onecut1 and Otx2, which bind to ThrbCRM1. The components of the gene regulatory networks that control the transition from multipotent to fate-restricted gene expression are not known. This work aims to identify and evaluate cis-regulatory elements proximal to Onecut1 to identify the gene regulatory networks involved in RPC fate-restriction.MethodWe identified regulatory elements through ATAC-seq and conservation, followed by reporter assays to screen for activity based on temporal and spatial criteria. The regulatory elements of interest were subject to deletion and mutation analysis to identify functional sequences and evaluated by quantitative flow cytometry assays. Finally, we combined the enhancer::reporter assays with candidate TF overexpression to evaluate the relationship between the TFs, the enhancers, and early vertebrate retinal development. Statistical tests included ANOVA, Kruskal-Wallis, or unpaired t-tests.ResultsTwo regulatory elements, ECR9 and ECR65, were identified to be active in ThrbCRM1(+) restricted RPCs. Candidate bHLH binding sites were identified as critical sequences in both elements. Overexpression of candidate bHLH TFs revealed specific enhancer-bHLH interactions. Nhlh1 overexpression expanded ECR65 activity into the Vsx2(+) RPC population, and overexpression of NeuroD1/NeuroG2/NeuroD4 had a similar effect on ECR9. Furthermore, bHLHs that were able to activate ectopic ECR9 reporter were able to induce endogenous Otx2 expression.ConclusionsThis work reports a large-scale screen to identify spatiotemporally specific regulatory elements near the Onecut1 locus. These elements were used to identify distinct populations in the developing retina. In addition, fate-restricted regulatory elements responded differentially to bHLH factors, and suggest a role for retinal bHLHs upstream of the Otx2 and Onecut1 genes during the formation of restricted RPCs from multipotent RPCs.

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GATA-targeted compounds modulate cardiac subtype cell differentiation in dual reporter stem cell line

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02259-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Mika J. Välimäki ◽

Robert S. Leigh ◽

Sini M. Kinnunen ◽

Alexander R. March ◽

Ana Hernández de Sande ◽

...

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

Cell Fate ◽

Reporter Gene ◽

Gene Regulatory Networks ◽

Regulatory Networks ◽

Cell Fate Decisions ◽

Dual Reporter ◽

Gene Regulatory ◽

Reporter Gene Assays

AbstractBackgroundPharmacological modulation of cell fate decisions and developmental gene regulatory networks holds promise for the treatment of heart failure. Compounds that target tissue-specific transcription factors could overcome non-specific effects of small molecules and lead to the regeneration of heart muscle following myocardial infarction. Due to cellular heterogeneity in the heart, the activation of gene programs representing specific atrial and ventricular cardiomyocyte subtypes would be highly desirable. Chemical compounds that modulate atrial and ventricular cell fate could be used to improve subtype-specific differentiation of endogenous or exogenously delivered progenitor cells in order to promote cardiac regeneration.MethodsTranscription factor GATA4-targeted compounds that have previously shown in vivo efficacy in cardiac injury models were tested for stage-specific activation of atrial and ventricular reporter genes in differentiating pluripotent stem cells using a dual reporter assay. Chemically induced gene expression changes were characterized by qRT-PCR, global run-on sequencing (GRO-seq) and immunoblotting, and the network of cooperative proteins of GATA4 and NKX2-5 were further explored by the examination of the GATA4 and NKX2-5 interactome by BioID. Reporter gene assays were conducted to examine combinatorial effects of GATA-targeted compounds and bromodomain and extraterminal domain (BET) inhibition on chamber-specific gene expression.ResultsGATA4-targeted compounds 3i-1000 and 3i-1103 were identified as differential modulators of atrial and ventricular gene expression. More detailed structure-function analysis revealed a distinct subclass of GATA4/NKX2-5 inhibitory compounds with an acetyl lysine-like domain that contributed to ventricular cells (%Myl2-eGFP+). Additionally, BioID analysis indicated broad interaction between GATA4 and BET family of proteins, such as BRD4. This indicated the involvement of epigenetic modulators in the regulation of GATA-dependent transcription. In this line, reporter gene assays with combinatorial treatment of 3i-1000 and the BET bromodomain inhibitor (+)-JQ1 demonstrated the cooperative role of GATA4 and BRD4 in the modulation of chamber-specific cardiac gene expression.ConclusionsCollectively, these results indicate the potential for therapeutic alteration of cell fate decisions and pathological gene regulatory networks by GATA4-targeted compounds modulating chamber-specific transcriptional programs in multipotent cardiac progenitor cells and cardiomyocytes. The compound scaffolds described within this study could be used to develop regenerative strategies for myocardial regeneration.

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Learning Large-Scale Fuzzy Cognitive Maps Based on Compressed Sensing and Application in Reconstructing Gene Regulatory Networks

IEEE Transactions on Fuzzy Systems ◽

10.1109/tfuzz.2017.2741444 ◽

2017 ◽

Vol 25 (6) ◽

pp. 1546-1560 ◽

Cited By ~ 16

Author(s):

Kai Wu ◽

Jing Liu

Keyword(s):

Compressed Sensing ◽

Gene Regulatory Networks ◽

Regulatory Networks ◽

Large Scale ◽

Cognitive Maps ◽

Fuzzy Cognitive Maps ◽

Gene Regulatory

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Large-Scale Statistical Inference of Gene Regulatory Networks: Local Network-Based Measures

Intelligent Systems Reference Library - Information Processing and Biological Systems ◽

10.1007/978-3-642-19621-8_8 ◽

2011 ◽

pp. 179-193

Author(s):

Frank Emmert-Streib

Keyword(s):

Statistical Inference ◽

Gene Regulatory Networks ◽

Regulatory Networks ◽

Large Scale ◽

Local Network ◽

Gene Regulatory

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

10.1101/2020.06.11.147314 ◽

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.

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rSNPBase 3.0: an updated database of SNP-related regulatory elements, element-gene pairs and SNP-based gene regulatory networks

Nucleic Acids Research ◽

10.1093/nar/gkx1101 ◽

2017 ◽

Vol 46 (D1) ◽

pp. D1111-D1116 ◽

Cited By ~ 15

Author(s):

Liyuan Guo ◽

Jing Wang

Keyword(s):

Gene Regulatory Networks ◽

Regulatory Networks ◽

Regulatory Elements ◽

Gene Pairs ◽

Gene Regulatory

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Mutations in Transcriptional Regulators Allow Selective Engineering of Signal Integration Logic

mBio ◽

10.1128/mbio.01171-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 4

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.

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Synthetic gene-regulatory networks in the opportunistic human pathogenStreptococcus pneumoniae

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1920015117 ◽

2020 ◽

Vol 117 (44) ◽

pp. 27608-27619 ◽

Cited By ~ 1

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.

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