Genome-Wide Forward Genetic Screens in Mouse ES Cells

2010 ◽  
pp. 217-242 ◽  
Author(s):  
Meng Amy Li ◽  
Stephen J. Pettitt ◽  
Kosuke Yusa ◽  
Allan Bradley
Keyword(s):  
Es Cells ◽  
Genetic Screens ◽  
Mouse Es Cells ◽  
Genome Wide ◽  
Forward Genetic Screens

scholarly journals Hoxa1 and TALE proteins display cross-regulatory interactions and form a combinatorial binding code on Hoxa1 targets

2016 ◽  
Author(s):  
Bony De Kumar ◽  
Hugo J. Parker ◽  
Ariel Paulson ◽  
Mark E. Parrish ◽  
Irina Pushel ◽  
...  
Keyword(s):  
Es Cells ◽  
Hox Proteins ◽  
Binding Motifs ◽  
Functional Roles ◽  
Regulatory Interactions ◽  
Mouse Es Cells ◽  
Genome Wide ◽  
A Genome ◽  
Combinatorial Interactions ◽  
Insight Into

AbstractHoxa1 has diverse functional roles in differentiation and development. We have identified and characterized properties of regions bound by Hoxa1 on a genome-wide basis in differentiating mouse ES cells. Hoxa1 bound regions are enriched for clusters of consensus binding motifs for Hox, Pbx and Meis and many display co-occupancy of Pbx and Meis. Pbx and Meis are members of the TALE family and genome-wide analysis of multiple TALE members (Pbx, Meis, TGIF, Prep1 and Prep2) show that nearly all Hoxa1 targets display occupancy of one or more TALE members. The combinatorial binding patterns of TALE proteins defines distinct classes of Hoxa1 targets and indicates a role as cofactors in modulating the specificity of Hox proteins. We also discovered extensive auto- and cross-regulatory interactions among the Hoxa1 and TALE genes. This study provides new insight into a regulatory network involving combinatorial interactions between Hoxa1 and TALE proteins.

scholarly journals TERRA regulate the transcriptional landscape of pluripotent cells through TRF1-dependent recruitment of PRC2

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Rosa María Marión ◽  
Juan J Montero ◽  
Isabel López de Silanes ◽  
Osvaldo Graña-Castro ◽  
Paula Martínez ◽  
...  
Keyword(s):  
Cell Fate ◽  
Es Cells ◽  
Epigenetic Changes ◽  
Pluripotent Cells ◽  
Telomere Repeat ◽  
Mouse Es Cells ◽  
Genome Wide ◽  
Binding Factor ◽  
Transcriptional Landscape ◽  
Pluripotency Genes

The mechanisms that regulate pluripotency are still largely unknown. Here, we show that Telomere Repeat Binding Factor 1 (TRF1), a component of the shelterin complex, regulates the genome-wide binding of polycomb and polycomb H3K27me3 repressive marks to pluripotency genes, thereby exerting vast epigenetic changes that contribute to the maintenance of mouse ES cells in a naïve state. We further show that TRF1 mediates these effects by regulating TERRA, the lncRNAs transcribed from telomeres. We find that TERRAs are enriched at polycomb and stem cell genes in pluripotent cells and that TRF1 abrogation results in increased TERRA levels and in higher TERRA binding to those genes, coincidental with the induction of cell-fate programs and the loss of the naïve state. These results are consistent with a model in which TRF1-dependent changes in TERRA levels modulate polycomb recruitment to pluripotency and differentiation genes. These unprecedented findings explain why TRF1 is essential for the induction and maintenance of pluripotency.

scholarly journals A simplified transposon mutagenesis method to perform phenotypic forward genetic screens in cultured cells

2019 ◽  
Author(s):  
Charlotte R. Feddersen ◽  
Lexy S. Wadsworth ◽  
Eliot Y. Zhu ◽  
Hayley R. Vaughn ◽  
Andrew P. Voigt ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Sequence Data ◽  
Sleeping Beauty ◽  
Transposon Mutagenesis ◽  
Loss Of Function ◽  
Genetic Screens ◽  
Sleeping Beauty Transposon ◽  
Genome Wide ◽  
Forward Genetic Screens ◽  
The Impact

AbstractThe introduction of genome-wide shRNA and CRISPR libraries has facilitated cell-based screens to identify loss-of-function mutations associated with a phenotype of interest. Approaches to perform analogous gain-of-function screens are less common, although some reports have utilized arrayed viral expression libraries or the CRISPR activation system. However, a variety of technical and logistical challenges make these approaches difficult for many labs to execute. In addition, genome-wide shRNA or CRISPR libraries typically contain of hundreds of thousands of individual engineered elements, and the associated complexity creates issues with replication and reproducibility for these methods. Here we describe a simple, reproducible approach using the Sleeping Beauty transposon system to perform phenotypic cell-based genetic screens. This approach employs only three plasmids to perform unbiased, whole-genome transposon mutagenesis. We also describe a ligation-mediated PCR method that can be used in conjunction with the included software tools to map raw sequence data, identify candidate genes associated with phenotypes of interest, and predict the impact of recurrent transposon insertions on candidate gene function. Finally, we demonstrate the high reproducibility of our approach by having three individuals perform independent replicates of a mutagenesis screen to identify drivers of vemurafenib resistance in cultured melanoma cells. Collectively, our work establishes a facile, adaptable method that can be performed by labs of any size to perform robust, genome-wide screens to identify genes that influence phenotypes of interest.

scholarly journals Applying CRISPR Screen in Diabetes Research

2021 ◽  
Author(s):  
Peng Yi ◽  
Noelle Morrow
Keyword(s):  
Cell Biology ◽  
Cancer Biology ◽  
Cell Types ◽  
Diabetes Research ◽  
Genetic Screens ◽  
Multiple Organs ◽  
Genome Wide ◽  
Forward Genetic Screens ◽  
Crispr Screen ◽  
Single Cell Type

The CRISPR/Cas9 genome editing system has been one of the greatest scientific discoveries in the last decade. The highly efficient and precise editing ability of this technology is of great therapeutic value and benefits the basic sciences as an advantageous research tool. In recent years, forward genetic screens utilizing CRISPR technology have been widely adopted, with genome-wide or pathway-focused screens leading to important and novel discoveries. CRISPR screens have been used primarily in cancer biology, virology and basic cell biology; but they have rarely been applied to diabetes research. A potential reason for this is that diabetes related research can be more complicated, often involving cross-talk between multiple organs or cell types. Nevertheless, many questions can still be reduced to the study of a single cell type if assays are carefully designed. Here we review the application of CRISPR screen technology and provide perspective on how it can be used in diabetes research.

scholarly journals A genome-wide knock-out screen for actors of epigenetic silencing reveals new regulators of germline genes and 2-cell like cell state

2021 ◽  
Author(s):  
Nikhil Gupta ◽  
Lounis Yakhou ◽  
Julien Richard Albert ◽  
Fumihito Miura ◽  
Laure Ferry ◽  
...  
Keyword(s):  
Es Cells ◽  
Epigenetic Mechanisms ◽  
Mammalian Development ◽  
Reporter System ◽  
Transcriptional Responses ◽  
Knock Out ◽  
Mouse Es Cells ◽  
Genome Wide ◽  
A Genome ◽  
Germline Genes

Epigenetic mechanisms are essential to establish and safeguard cellular identities in mammals. They dynamically regulate the expression of genes, transposable elements, and higher-order chromatin structures. Expectedly, these chromatin marks are indispensable for mammalian development and alterations often lead to diseases such as cancer. Molecularly, epigenetic mechanisms rely on factors to establish patterns, interpret them into a transcriptional output, and maintain them across cell divisions. A global picture of these phenomena has started to emerge over the years, yet many of the molecular actors remain to be discovered. In this context, we have developed a reporter system sensitive to epigenetic perturbations to report on repressive pathways based on Dazl, which is normally repressed in mouse ES cells. We used this system for a genome-wide CRISPR knock-out screen, which yielded expected hits (DNMT1, UHRF1, MGA), as well as novel candidates. We prioritized the candidates by secondary screens, and led further experiments on 6 of them: ZBTB14, KDM5C, SPOP, MCM3AP, BEND3, and KMT2D. Our results show that all 6 candidates regulate the expression of germline genes. In addition, we find that removal of ZBTB14, KDM5C, SPOP and MCM3AP led to similar transcriptional responses, including a reactivation of the 2-cell like cell (2CLC) signature. Therefore, our genetic screen has identified new regulators of key cellular states.

scholarly journals Applying CRISPR Screen in Diabetes Research

2021 ◽  
Author(s):  
Peng Yi ◽  
Noelle Morrow
Keyword(s):  
Cell Biology ◽  
Cancer Biology ◽  
Cell Types ◽  
Diabetes Research ◽  
Genetic Screens ◽  
Multiple Organs ◽  
Genome Wide ◽  
Forward Genetic Screens ◽  
Crispr Screen ◽  
Single Cell Type

The CRISPR/Cas9 genome editing system has been one of the greatest scientific discoveries in the last decade. The highly efficient and precise editing ability of this technology is of great therapeutic value and benefits the basic sciences as an advantageous research tool. In recent years, forward genetic screens utilizing CRISPR technology have been widely adopted, with genome-wide or pathway-focused screens leading to important and novel discoveries. CRISPR screens have been used primarily in cancer biology, virology and basic cell biology; but they have rarely been applied to diabetes research. A potential reason for this is that diabetes related research can be more complicated, often involving cross-talk between multiple organs or cell types. Nevertheless, many questions can still be reduced to the study of a single cell type if assays are carefully designed. Here we review the application of CRISPR screen technology and provide perspective on how it can be used in diabetes research.

scholarly journals Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Iris Jonkers ◽  
Hojoong Kwak ◽  
John T Lis
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Half Life ◽  
Es Cells ◽  
Negative Effects ◽  
Pol Ii ◽  
Crucial Step ◽  
Mouse Es Cells ◽  
Genome Wide ◽  
Positive Effect

Production of mRNA depends critically on the rate of RNA polymerase II (Pol II) elongation. To dissect Pol II dynamics in mouse ES cells, we inhibited Pol II transcription at either initiation or promoter-proximal pause escape with Triptolide or Flavopiridol, and tracked Pol II kinetically using GRO-seq. Both inhibitors block transcription of more than 95% of genes, showing that pause escape, like initiation, is a ubiquitous and crucial step within the transcription cycle. Moreover, paused Pol II is relatively stable, as evidenced from half-life measurements at ∼3200 genes. Finally, tracking the progression of Pol II after drug treatment establishes Pol II elongation rates at over 1000 genes. Notably, Pol II accelerates dramatically while transcribing through genes, but slows at exons. Furthermore, intergenic variance in elongation rates is substantial, and is influenced by a positive effect of H3K79me2 and negative effects of exon density and CG content within genes.

scholarly journals Genome-wide Screens Reveal Escherichia Coli Genes Required for Growth of T1-like Phage LL5 and rV5-like Phage LL12

2018 ◽  
Author(s):  
Denish Piya ◽  
Lauren Lessor ◽  
Brian Koehler ◽  
Ashley Stonecipher ◽  
Jesse Cahill ◽  
...  
Keyword(s):  
Inner Core ◽  
Essential Gene ◽  
Outer Core ◽  
Genetic Screens ◽  
Gene Deletions ◽  
Factors Affecting ◽  
E Coli ◽  
Genome Wide ◽  
Forward Genetic Screens ◽  
Potential Use

Factors affecting the host-virus interaction must be understood for the effective application of bacteriophages to combat bacterial pathogens. Two novel E. coli phages, the T1-like siphophage LL5 and the rV5-like myophage LL12, were subjected to forward genetic screens against the Keio collection, a library of single non-essential gene deletions in E.coli str. BW25113. These genome-wide screens and subsequent experiments identified eight genes required for efficient propagation of phage LL5 and six genes required for propagation of LL12. The majority of the genes identified were involved in production of the phage receptors. E. coli mutants deficient in heptose II and the phosphoryl substituent of heptose I of the inner core lipopolysaccharide (LPS) were unable to propagate phage LL5, as were mutants deficient in the outer membrane protein TolC. Mutants lacking glucose I of the LPS outer core failed to propagate LL12. Two cytoplasmic chaperones, PpiB and SecB, were found to be required for efficient propagation of phage LL5 but not LL12. This approach may be useful for identifying phage receptors and required host factors in other phages, which would provide valuable information for their potential use as therapeutics and for phage engineering.

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