Nod1-dependent NF-kB activation initiates hematopoietic stem cell specification in response to small Rho GTPases
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ABSTRACT:
PROVIDER: PRJNA932255 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR23360301_1.fastq.gz | Fastqsanger.gz | |||
| SRR23360301_2.fastq.gz | Fastqsanger.gz | |||
| SRR23360302_1.fastq.gz | Fastqsanger.gz | |||
| SRR23360302_2.fastq.gz | Fastqsanger.gz | |||
| SRR23360303_1.fastq.gz | Fastqsanger.gz |
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Project description:The possibility of specifying functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (hPSCs) would overcome current limitations related to HSPC transplantation. However, generating hPSC-derived HSPCs has been elusive, necessitating a better understanding of the native developmental mechanisms that trigger HSPC specification. Here, we revealed in vivo an intrinsic inflammatory mechanism triggered by Nod1 that drives early hemogenic endothelium (HE) patterning to specify HSPCs. Our genetic and chemical experiments showed that HSPCs failed to specify in the absence of Nod1 and its downstream kinase Ripk2. Rescue experiments demonstrated that Nod1 and Ripk2 acted through NF-kB, and that small Rho GTPases are at the apex of this mechanism. Manipulation of NOD1 in a human system of hPSCs differentiation towards the definitive hematopoietic lineage indicated functional conservation. This work establishes the RAC1-NOD1-RIPK2-NFkB axis as the earliest inflammatory inductor that intrinsically primes the HE for proper HSPC specification. Manipulation of this pathway could help derive a competent HE amenable to specify functional patient specific HSPCs for the treatment of blood disorders. Keywords: NOD1, RIPK2, NF-kB, RAC1, Rho GTPases, Hematopoietic Stem and Progenitor Cell Specification, Hemogenic Endothelium.
2023-10-26 | GSE224708 | GEO
Project description:Uncovering the mechanisms regulating hematopoietic specification not only would overcome current limitations related to hematopoietic stem and progenitor cell (HSPC) transplantation, but also advance cellular immunotherapies. However, generating functional human induced pluripotent stem cell (hiPSC)-derived HSPCs and their derivatives has been elusive, necessitating a better understanding of the developmental mechanisms that trigger HSPC specification. Here, we reveal that early activation of the Nod1-Ripk2-NF-kB inflammatory pathway in endothelial cells (ECs) primes them to switch fate towards definitive hemogenic endothelium, a pre-requisite to specify HSPCs. Our genetic and chemical embryonic models show that HSPCs fail to specify in the absence of Nod1 and its downstream kinase Ripk2 due to a failure on hemogenic endothelial (HE) programming, and that small Rho GTPases coordinate the activation of this pathway. Manipulation of NOD1 in a human system of definitive hematopoietic differentiation indicates functional conservation. This work establishes the RAC1-NOD1-RIPK2-NF-kB axis as a critical intrinsic inductor that primes ECs prior to HE fate switch and HSPC specification. Manipulation of this pathway could help derive a competent HE amenable to specify functional patient specific HSPCs and their derivatives for the treatment of blood disorders.
| S-EPMC10667254 | biostudies-literature
Project description:We have used microarray technology to identify the transcriptional targets of Rho subfamily GTPases. This analysis indicated that murine fibroblasts transformed by these proteins show similar transcriptomal profiles. Functional annotation of the regulated genes indicate that Rho subfamily GTPases target a wide spectrum of biological functions, although loci encoding proteins linked to proliferation and DNA synthesis/transcription are up-regulated preferentially. Rho proteins promote four main networks of interacting proteins nucleated around E2F, c-Jun, c-Myc, and p53. Of those, E2F, c-Jun and c-Myc are essential for the maintenance of cell transformation. Inhibition of Rock, one of the main Rho GTPase targets, leads to small changes in the transcriptome of Rho-transformed cells. Rock inhibition decreases c-myc gene expression without affecting the E2F and c-Jun pathways. Loss-of-function studies demonstrate that c-Myc is important for the blockage of cell-contact inhibition rather than for promoting the proliferation of Rho-transformed cells. However, c-Myc overexpression does not bypass the inhibition of cell transformation induced by Rock blockage, indicating that c-Myc is essential, but not sufficient, for Rock-dependent transformation. These results reveal the complexity of the genetic program orchestrated by the Rho subfamily and pinpoint protein networks that mediate different aspects of the malignant phenotype of Rho-transformed cells Keywords: Rho/Rac GTPases, microarray, oncogenesis, proliferation, gene expression, transcription, Rock, c-Myc
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Project description:We performed CBX3 knockdown in lung adenocarcinoma cells along with RNA sequencing. The expression profiling showed Rho GTPases signaling as a potential CBX3 target and the conclusions were also verified in clinical samples.
2021-05-05 | GSE173858 | GEO
Project description:We have used microarray technology to identify the transcriptional targets of Rho subfamily GTPases. This analysis indicated that murine fibroblasts transformed by these proteins show similar transcriptomal profiles. Functional annotation of the regulated genes indicate that Rho subfamily GTPases target a wide spectrum of biological functions, although loci encoding proteins linked to proliferation and DNA synthesis/transcription are up-regulated preferentially. Rho proteins promote four main networks of interacting proteins nucleated around E2F, c-Jun, c-Myc, and p53. Of those, E2F, c-Jun and c-Myc are essential for the maintenance of cell transformation. Inhibition of Rock, one of the main Rho GTPase targets, leads to small changes in the transcriptome of Rho-transformed cells. Rock inhibition decreases c-myc gene expression without affecting the E2F and c-Jun pathways. Loss-of-function studies demonstrate that c-Myc is important for the blockage of cell-contact inhibition rather than for promoting the proliferation of Rho-transformed cells. However, c-Myc overexpression does not bypass the inhibition of cell transformation induced by Rock blockage, indicating that c-Myc is essential, but not sufficient, for Rock-dependent transformation. These results reveal the complexity of the genetic program orchestrated by the Rho subfamily and pinpoint protein networks that mediate different aspects of the malignant phenotype of Rho-transformed cells Experiment Overall Design: In order to generate the cell clones used in this study, we took advantage of the oncogenic properties of the constitutively active versions (Q63L mutants) of Rho subfamily proteins when overexpressed in rodent fibroblasts (Schuebel et al., 1998). Based on this property, we transfected NIH3T3 cells with plasmids encoding the indicated versions of Rho subfamily proteins to obtain foci of transformed cells. Selected foci were picked, pooled, and used for the subsequent microarray experiments. Experiment Overall Design: To avoid the activation of genetic programs related to serum withdrawal or contact inhibition that may confound the detection of Rho-specific transcriptomal changes (Coller et al., 2006), we cultured the chosen cell lines and the parental NIH3T3 cells in the presence of serum and maintained them at confluency levels lower than 70% prior to RNA extraction. In addition, we isolated total RNAs from eight (in the case of NIH3T3 cells), seven (in the case of RhoAQ63L-transformed cells) and six (in the case of RhoBQ63L- and RhoCQ63L-transformed cells) independent cell cultures in order to make it possible a robust statistical treatment of the data obtained. Experiment Overall Design: three 10-cm diameter plates containing exponentially growing cultures of IMB11-1P (expressing RhoAQ63L) IMB11-2P (expressing RhoBQ63L), or IMB11-3P (expressing RhoCQ63L) cells were washed with PBS and their total cellular RNAs isolated using the RNeasy kit (Qiagen) according to the supplierâs specifications. The quantity and quality of the total RNAs obtained was determined using 6000 Nano Chips (Agilent Technologies). Total RNA samples (4 ug) were then processed for hybridization on MGU75Av2 microarrays (Affymetrix) using standard Affymetrix protocols at the CIC Genomics and Proteomics Facility (www.cicancer.org). Normalization, filtering and analysis of the raw data obtained from the microarrays was carried out with the Bioconductor software (www.bioconductor.com) using de ReadAffy package and the RMA application. The RMA algorithm was selected over the standard Affymetrix software because it provides a better precision in signal detection to achieve adequate normalization of multiple microarray hybridizations, especially in cases of low levels of gene expression (Bolstad et al., 2004; Gautier et al., 2004; Gentleman et al., 2004; Parrish & Spencer, 2004). We considered a gene to be differentially expressed when exhibiting a signal ⥠100 and met the following criteria: in the case of the characterization of the transcriptome of Rho-transformed cells, we regarded a gene as common to all GTPases when: i) It showed a fold change ⥠1.5 in at least two of the cell lines used. ii) The fold change in the third cell line was ⥠1.0 and displayed a similar variation trend when compared to the other two cell lines (i.e., similar up-regulation or down-modulation in the three cell lines). iii) The fold change values in the three cell lines had always P values ⤠0.01. We regarded a gene as common to only two GTPases when: i) It showed a fold change ⥠1.5 in two the cell lines with P values ⤠0.01. ii) The fold change in the third cell line was non-existent or, alternatively, had P values ⥠0.01. We considered a gene as uniquely-regulated by a GTPase when: i) The fold change in the expression levels of is transcript in the cell line transformed by that GTPase was ⥠1.5 with P value ⤠0.01. ii) The fold change, if any, obtained in the other cell lines had P values ⥠0.01. Statistical analyses were performed using F-statistics.
2008-06-14 | E-GEOD-5913 | biostudies-arrayexpress
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2016-12-23 | PXD004190 | Pride
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