Dissecting Molecular Phenotypes Through FACS-Based Pooled CRISPR Screens

Genolet, Oriana; Lustig, Liat Ravid; Schulz, Edda G.

doi:10.1007/7651_2021_457

Cited by 8 publications

(6 citation statements)

References 36 publications

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“…This may also be reflected in the inferred gene regulatory network, in which we observe an inhibitory influence of FOS and EGR1 on multiple immediate early transcription factors and immediate early genes. To further decipher the complex feedback regulation between the transcription network and signal transduction, perturbation datasets that additionally measure the activity of signaling are required (Dessauges et al , 2022; Genolet et al , 2022).…”

Section: Discussionmentioning

confidence: 99%

Targeted Perturb-seq Reveals EGR1 and FOS as Key Regulators of the Transcriptional RAF-MAPK Response

Kassem,

Sieber,

Klinger

et al. 2024

Preprint

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The MAPK pathway is an important cellular signaling cascade whose dysregulation causes a variety of diseases. While the upstream regulators of this cascade have been extensively characterized, the understanding of how its activation translates into different transcriptional responses remains poorly understood. This study attempts to fill this knowledge gap by using targeted Perturb-seq against 22 transcription factors in an inducible model system for RAF-MAPK signaling. A topology-based modeling approach is applied to the obtained data to construct a directional interaction network. By removing coherent feed-forward loops and integrating the expression kinetics of transcription factors, a parsimonious network structure is derived that distinguishes direct from indirect interactions between the investigated transcription factors and their targets. In particular, EGR1 and FOS are found to act as orthogonal upstream regulators of the RAF-MAPK response. The results presented here provide valuable insights into the organization of the transcriptional network downstream of RAF-MAPK signaling and thus provide a basis for a better understanding of this complex process.

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Section: Discussionmentioning

confidence: 99%

Targeted Perturb-seq Reveals EGR1 and FOS as Key Regulators of the Transcriptional RAF-MAPK Response

Kassem,

Sieber,

Klinger

et al. 2024

Preprint

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“…Phenotypic enrichment based on RNA levels was performed as previously described 90 . The PrimeFlow RNA assay (Thermo Fisher) was used as described above.…”

Section: Methodsmentioning

confidence: 99%

GATA transcription factors drive initial Xist upregulation after fertilization through direct activation of long-range enhancers

Ravid Lustig,

Sampath Kumar,

Schwämmle

et al. 2023

Nat Cell Biol

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X-chromosome inactivation (XCI) balances gene expression between the sexes in female mammals. Shortly after fertilization, upregulation of Xist RNA from one X chromosome initiates XCI, leading to chromosome-wide gene silencing. XCI is maintained in all cell types, except the germ line and the pluripotent state where XCI is reversed. The mechanisms triggering Xist upregulation have remained elusive. Here we identify GATA transcription factors as potent activators of Xist. Through a pooled CRISPR activation screen in murine embryonic stem cells, we demonstrate that GATA1, as well as other GATA transcription factors can drive ectopic Xist expression. Moreover, we describe GATA-responsive regulatory elements in the Xist locus bound by different GATA factors. Finally, we show that GATA factors are essential for XCI induction in mouse preimplantation embryos. Deletion of GATA1/4/6 or GATA-responsive Xist enhancers in mouse zygotes effectively prevents Xist upregulation. We propose that the activity or complete absence of various GATA family members controls initial Xist upregulation, XCI maintenance in extra-embryonic lineages and XCI reversal in the epiblast.

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“…SgRNAs designed for CRISPR-Cas9 deletions were cloned into the pSpCas9(BB)-2A-GFP vector (Addgene #48138) or into the pSpCas9(BB)-2A-mCherry vector as previously described 41 . SgRNAs designed for CRISPRi experiments targeting the JPX promoter were cloned into the PB_rtTA_BsmBI vector (Addgene, #126028) and sgRNAs targeting Enh5 were cloned into a multiexpression sgRNA vector, a gift from the Schulz lab 90 . All sgRNA sequences can be found in Supplementary Table1 for rhesus macaque and Table2 for marmoset.…”

Section: Methodsmentioning

confidence: 99%

Extensive remodelling ofXISTregulatory networks during primate evolution

Cazottes,

Alfeghaly,

Rognard

et al. 2023

Preprint

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Unravelling how gene regulatory networks are remodelled during evolution is crucial to understand how species adapt to environmental changes. We addressed this question for X-chromosome inactivation, a process essential to female development that is governed, in eutherians, by theXISTlncRNA and itscis-regulators. To reach high resolution, we studied closely related primate species, spanning 55 million years of evolution. We show that theXISTregulatory circuitry has diversified extensively over such evolutionary timeframe. The insertion of a HERVK transposon has reshuffledXIST3D interaction network in macaque embryonic stem cells (ESC) andXISTexpression is maintained by the additive effects of theJPXlncRNA gene and a macaque specific enhancer. In contrast,JPXis the main contributor toXISTexpression in human ESCs but is not significantly involved inXISTregulation in marmoset ESCs. None of these entities are however under purifying selection, which suggests that neutrally evolving non-coding elements harbour high adaptive potentials.

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Dissecting Molecular Phenotypes Through FACS-Based Pooled CRISPR Screens

Cited by 8 publications

References 36 publications

Targeted Perturb-seq Reveals EGR1 and FOS as Key Regulators of the Transcriptional RAF-MAPK Response

Targeted Perturb-seq Reveals EGR1 and FOS as Key Regulators of the Transcriptional RAF-MAPK Response

GATA transcription factors drive initial Xist upregulation after fertilization through direct activation of long-range enhancers

Extensive remodelling ofXISTregulatory networks during primate evolution

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