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Expression data from WT and Elektra (Slfn2 mutant, loss of function) homozygous hematopoietic multipotent progenitor cells extracted from mice.

ABSTRACT: Expression data from WT and Elektra (Slfn2 mutant, loss of function) homozygous hematopoietic multipotent progenitor cells extracted from mice.

PROVIDER: PRJNA929934 | ENA |

REPOSITORIES: ENA

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Project description:In this study we investigated the effect of Slfn2 loss of function in the murine hematopoietic system. We chose to evaluate gene expression differences in multipotent progenitors, as this population was found to be diminshed in elektra homozygous mice, additionally the MPP population also displayed increased cell cycling compared to wildtype and increased apoptosis. Abstract from associated publication copied in below, from Warsi et al. 2022 Schlafen2 is a regulator of quiescence in adult murine hematopoietic stem cells, published in Haematologica. DOI 10.3324/haematol.2021.279799 Even though hematopoietic stem cells (HSCs) are characterized by their ability to self-renew and differentiate, they primarily reside in quiescence. Despite the immense importance of this quiescent state, its maintenance and regulation is still incompletely understood. Schlafen2 (Slfn2) is a cytoplasmic protein known to be involved in cell proliferation, differentiation, quiescence, interferon response, and regulation of the immune system. Interestingly, Slfn2 is highly expressed in primitive hematopoietic cells. To investigate the role of Slfn2 in the regulation of HSCs we have studied HSC function in the elektra mouse model, where the elektra allele of the Slfn2 gene contains a point mutation causing loss of function of the Slfn2 protein. We found that homozygosity for the elektra allele caused a decrease of primitive hematopoietic compartments in murine bone marrow. We further found that transplantation of elektra bone marrow and purified HSCs resulted in a significantly reduced regenerative capacity of HSCs in competitive transplantation settings. Importantly, we found that a significantly higher fraction of elektra HSCs (as compared to WT HSCs) were actively cycling, suggesting that the mutation in Slfn2 increases HSC proliferation. This additionally caused an increased amount of apoptotic stem and progenitor cells. Taken together, our findings demonstrate that dysregulation of Slfn2 results in a functional deficiency of primitive hematopoietic cells, which is particularly reflected by a drastically impaired ability to reconstitute the hematopoietic system following transplantation and an increase in HSC proliferation. This study thus identifies Slfn2 as a novel and critical regulator of a dult HSCs and HSC quiescence.

Project description:During adult bone marrow hematopoiesis, extremely rare and dormant hematopoietic stem cells (HSCs) harbor the highest self-renewal activity within all blood cells. They give rise to active HSCs, which generate multipotent progenitors (MPPs) which differentiate into lineage-committed progenitors and subsequently mature cells. While HSCs are characterized by long-term self-renewal capacity, quiescence and multipotency, MPPs show steadily decreasing self-renewal activity, are cycling but are thought to maintain multipotency. To establish a comprehensive genome-wide landscape of expressed transcripts, we performed a quantitative transcriptome analysis by next-generation sequencing (RNA-seq) of seven ex vivo FACS-sorted mouse HSC/progenitor populations. Eleven-fold coverage of the genome was achieved, revealing quantification of > 27,000 mRNA species of which 589 long non-coding RNAs (lncRNAs) were quantified. A profile of 79 differentially expressed lncRNAs in HSC-MPPs was identified suggesting a role for these RNA species in HSC/progenitor biology. Expression clusters of transcription factors and cell adhesion molecules are identified between the different cell populations. Dormant HSCs, as identified by label-retaining assays, showed a highly differential expression profile compared to active HSCs. In addition to >200 differentially expressed cell surface receptors and lncRNAs, processes including metabolism, development, immune response, signaling (TGFb, Kit, senescence/autophagy) are distinct between the two types of HSCs. In addition, using whole cell proteome analysis of FACS-sorted HSCs and MPP1, >6,000 proteins were identified by quantitative tandem mass spectrometry. Quantification of these proteins confirmed the close relationship between these cell types also seen in their transcript profile and revealed processes such as energy metabolism, immune response and cell cycle to be modulated along early lineage progression. While MPP1/2 still show multilineage potential in reconstitution experiments, a strong lineage bias and low self-renewal potential is observed in mice reconstituted with MPP3/4. These functional differences are accompanied by complex changes in their transcriptome and is also revealed by principal component analysis. In summary, the global mRNA, lncRNA and proteome signatures uncovered here and which are complemented by functional assays, provide a comprehensive and searchable resource of the molecular make-up of the entire HSC/progenitor population present in the bone marrow. These data will provide the basis for a global understanding of stem cell biology in the adult blood system. The uploaded dataset corresponds to the quantitative proteomic comparison of HSC and MPP1, which was done in three biological replicates. Data analysis: MS raw data files were processed with MaxQuant (version 1.3.0.5) (Cox and Mann 2008). Enzyme specificity was set to trypsin/P and a maximum of two missed cleavages were allowed. Cysteine carbamidomethylation and methionine oxidation were selected as fixed and variable modifications, respectively. The derived peak list was searched using the built-in Andromeda search engine (version 1.3.0.5) in MaxQuant against the Uniprot mouse database (2013.02.20) containing 75,721 proteins to which 247 frequently observed contaminants as well as reversed sequences of all entries had been added. Initial maximal allowed mass tolerance was set to 20 ppm for peptide masses, followed by 6 ppm in the main search, and 0.5 Dalton for fragment ion masses. The minimum peptide length was set to six amino acid residues and three labeled amino acid residues were allowed. A 1% false discovery rate (FDR) was required at both the protein level and the peptide level. In addition to the FDR threshold, proteins were considered identified if they had at least one unique peptide. The protein identification was reported as an indistinguishable “protein group” if no unique peptide sequence to a single database entry was identified. The ‘match between runs’ was enabled for consecutive peptide fractions with a 2 minutes time window. The iBAQ algorithm was used for estimation of the abundance of different proteins within a single sample (proteome) (Schwanhausser 2011). For evaluation of differential protein expression between HSC and MPP1, statistical analysis was performed for the proteins quantified in all three replicates using the Limma package in R/Bioconductor (Gentleman 2004, Smyth 2004). After fitting a linear model to the data, an empirical Bayes moderated t-test was used for the protein ratios, which were weighted on log10(summed peptide intensities) in order to capture the effect that the statistical spread of unregulated proteins is much more focused for highly abundant proteins than for low abundance ones (Cox 2008). P-values were then adjusted for multiple testing with Benjamini and Hochberg's method and proteins with an adjusted p-value lower than 0.1 were considered to be differentially expressed between HSC and MPP1. Associated transcriptomics data has been deposited at ArrayExpress with accession E-MTAB-2262.

Dataset Information

Expression data from WT and Elektra (Slfn2 mutant, loss of function) homozygous hematopoietic multipotent progenitor cells extracted from mice.

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