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Background Hematopoietic stem cells (HSCs) are the guarantor of the proper functioning of hematopoiesis due to their incredible diversity of potential. During aging, heterogeneity of HSCs changes, contributing to the deterioration of the immune system. In this study, we revisited mouse HSC compartment and its transcriptional plasticity during aging at unicellular scale. Results Through the analysis of 15,000 young and aged transcriptomes, we identified 15 groups of HSCs revealing rare and new specific HSC abilities that change with age. The implantation of new trajectories complemented with the analysis of transcription factor activities pointed consecutive states of HSC differentiation that were delayed by aging and explained the bias in differentiation of older HSCs. Moreover, reassigning cell cycle phases for each HSC clearly highlighted an imbalance of the cell cycle regulators of very immature aged HSCs that may contribute to their accumulation in an undifferentiated state. Conclusions Our results establish a new reference map of HSC differentiation in young and aged mice and reveal a potential mechanism that delays the differentiation of aged HSCs and could promote the emergence of age-related hematologic diseases.

Autophagy is an essential cellular process that maintains homeostasis by recycling damaged organelles and nutrients during development and cellular stress. ZKSCAN3 is the sole identified master transcriptional repressor of autophagy in human cell lines. How ZKSCAN3 achieves autophagy repression at the mechanistic or organismal level however still remains to be elucidated. Furthermore, Zkscan3 knockout mice display no discernable autophagy-related phenotypes, suggesting that there may be substantial differences in the regulation of autophagy between normal tissues and tumor cell lines. Here, we demonstrate that vertebrate ZKSCAN3 and Drosophila M1BP are functionally homologous transcription factors in autophagy repression. Expression of ZKSCAN3 in Drosophila prevents premature autophagy onset due to loss of M1BP function and conversely, M1BP expression in human cells can prevent starvation-induced autophagy due to loss of nuclear ZKSCAN3 function. In Drosophila ZKSCAN3 binds genome-wide to sequences targeted by M1BP and transcriptionally regulates the majority of M1BP-controlled genes, demonstrating the evolutionary conservation of the transcriptional repression of autophagy. This study thus  allows the potential for transitioning the mechanisms, gene targets and plethora metabolic processes controlled by M1BP onto ZKSCAN3 and opens up Drosophila as a tool in studying the function of ZKSCAN3 in autophagy and tumourigenesis.

Single-cell transcriptomic technologies enable the uncovering and characterization of cellular heterogeneity and pave the way for studies aiming at understanding the origin and consequences of it. The hematopoietic system is in essence a very well adapted model system to benefit from this technological advance because it is characterized by different cellular states. Each cellular state, and its interconnection, may be defined by a specific location in the global transcriptional landscape sustained by a complex regulatory network. This transcriptomic signature is not fixed and evolved over time to give rise to less efficient hematopoietic stem cells (HSC), leading to a well-documented hematopoietic aging. Here, we review the advance of single-cell transcriptomic approaches for the understanding of HSC heterogeneity to grasp HSC deregulations upon aging. We also discuss the new bioinformatics tools developed for the analysis of the resulting large and complex datasets. Finally, since hematopoiesis is driven by fine-tuned and complex networks that must be interconnected to each other, we highlight how mathematical modeling is beneficial for doing such interconnection between multilayered information and to predict how HSC behave while aging.

An amendment to this paper has been published and can be accessed via the original article.

Bone marrow (BM) produces all blood and immune cells deriving from hematopoietic stem cells (HSCs). The decrease of immune cell production during aging is one of the features of immunosenescence. The impact of redox dysregulation in BM aging is still poorly understood. Here we use TP53INP1-deficient (KO) mice endowed with chronic oxidative stress to assess the influence of aging-associated redox alterations in BM homeostasis. We show that TP53INP1 deletion has no impact on aging-related accumulation of HSCs. In contrast, the aging-related contraction of the lymphoid compartment is mitigated in TP53INP1 KO mice. B cells that accumulate in old KO BM are differentiating cells that can mature into functional B cells. Importantly, this phenotype results from B cell-intrinsic events associated with defective redox control. Finally, we show that oxidative stress in aged TP53INP1-deficient mice maintains STAT5 expression and activation in early B cells, driving high Pax5 expression, which provides a molecular mechanism for maintenance of B cell development upon aging.

Classical biochemical and molecular methods for discerning cells with epigenetic modifications are often biologically perturbing or even destructive. We wondered whether the noninvasive laser tweezer Raman spectroscopy technique allowed the discrimination of single living human cells undergoing epigenetic modifications. Human Jurkat leukemic cells were treated with inhibitors of histone deacetylases (trichostatin A and MS-275). Epigenetic changes were monitored through histone electrophoresis, nuclear image cytometry and laser tweezer Raman spectroscopy. Treatment of Jurkat cells with histone deacetylase inhibitors increased histone acetylation and induced chromatin organization changes. Characteristic vibrations, issued from laser tweezer Raman spectroscopy analyses, mostly assigned to DNA and proteins allowed discerning histone deacetylase inhibitor-treated cells from control with high confidence. Statistical processing of laser tweezer Raman spectroscopy data led to the definition of specific biomolecular fingerprints of each cell group. This original study shows that laser tweezer Raman spectroscopy is a label-free rapid tool to identify living cells that underwent epigenetic changes.

Single-cell transcriptomic technologies enable the uncovering and characterization of cellular heterogeneity and pave the way for studies aiming at understanding the origin and consequences of it. The hematopoietic system is in essence a very well adapted model system to benefit from this technological advance because it is characterized by different cellular states. Each cellular state, and its interconnection, may be defined by a specific location in the global transcriptional landscape sustained by a complex regulatory network. This transcriptomic signature is not fixed and evolved over time to give rise to less efficient hematopoietic stem cells (HSC), leading to a well-documented hematopoietic aging. Here, we review the advance of single-cell transcriptomic approaches for the understanding of HSC heterogeneity to grasp HSC deregulations upon aging. We also discuss the new bioinformatics tools developed for the analysis of the resulting large and complex datasets. Finally, since hematopoiesis is driven by fine-tuned and complex networks that must be interconnected to each other, we highlight how mathematical modeling is beneficial for doing such interconnection between multilayered information and to predict how HSC behave while aging.

Significance Reactive oxygen species (ROS) play a role in signaling in immune cells, in particular in B cell activation and terminal differentiation in secondary lymphoid organs. Nevertheless, their role in B cell development in the bone marrow (BM) still remains poorly explored. TP53INP1 is a target of the tumor suppressor p53, which mediates its antioxidant activity. We report the surprising observation that chronic oxidative stress in TP53INP1-deficient mice rescues B lymphopoiesis in the BM during aging. ROS sustain IL-7R signaling in aged TP53INP1-deficient BM through maintenance of active STAT5 transcription factor, driving the expression of the B lineage Pax5 transcription factor. This work suggests that antioxidants cannot be in favor of antibody-producing cell development.

Background The epigenetic machinery is frequently altered in acute myeloid leukemia. Focusing on cytogenetically normal (CN) AML, we previously described an abnormal H3K27me3 enrichment covering 70 kb on the HIST1 cluster (6.p22) in CN-AML patient blasts. Here, we further investigate the molecular, functional, and prognosis significance of this epigenetic alteration named H3K27me3 HIST1 in NPM1 -mutated ( NPM1 mut) CN-AML. Results We found that three quarter of the NPM1 mut CN-AML patients were H3K27me3 HIST1 high . H3K27me3 HIST1 high group of patients was associated with a favorable outcome independently of known molecular risk factors. In gene expression profiling, the H3K27me3 HIST1 high mark was associated with lower expression of the histone genes HIST1H1D , HIST1H2BG , HIST1H2AE , and HIST1H3F and an upregulation of genes involved in myelomonocytic differentiation. Mass spectrometry analyses confirmed that the linker histone protein H1d, but not the other histone H1 subtypes, was downregulated in the H3K27me3 HIST1 high group of patients. H1d knockdown primed ATRA-mediated differentiation of OCI-AML3 and U937 AML cell lines, as assessed on CD11b/CD11c markers, morphological and gene expression analyses. Conclusions Our data suggest that NPM1 mut AML prognosis depends on the epigenetic silencing of the HIST1 cluster and that, among the H3K27me3 silenced histone genes, HIST1H1D plays a role in AML blast differentiation. Graphical abstract

Hematopoietic stem cell (HSC) aging is a multifactorial event that leads to changes in HSC properties and function. These changes are intrinsically coordinated and affect the early hematopoiesis, involving hematopoietic stem and progenitor cells (HSPCs). The objective of this work is to better understand the mechanisms and factors controlling these changes. We have therefore developed an original strategy to construct a Boolean network of genes explaining the priming and homeostasis of HSCs (graphical abstract). Based on our previous scRNA-seq data, we performed an exhaustive analysis of the transcriptional network and identified active transcription modules or regulons along the differentiation trajectory of selected HSPC states. This global view of transcriptional regulation led us to focus on 15 components, 13 selected TFs (Tal1, Fli1, Gata2, Gata1, Zfpm1, Egr1, Junb, Ikzf1, Myc, Cebpa, Bclaf1, Klf1, Spi1) and 2 complexes regulating the ability of HSC to cycle (CDK4/6 - Cyclin D and CIP/KIP). We then defined the connections controlling the differentiation dynamics of HSC states and constructed an influence graph between the TFs involved in the dynamics by mixing observations from our scRNA-seq data and knowledge from the literature. Then, using answer set programming (ASP) and in silico perturbation analysis, we obtained a Boolean model which is the solution of a Boolean satisfiability problem. Finally, perturbation of the model based on age-related changes revealed important regulations, such as the overactivation of Egr1 and Junb or the loss of Cebpa activation by Gata2, which were found to be relevant for the myeloid bias of aged HSC. Our work shows the efficiency of the combination of manual and systematic methods to elaborate a Boolean model. The developed strategy led to the proposal of new regulatory mechanisms underlying the differentiation bias of aged HSCs, explaining the decreased transcriptional priming of HSCs to all mature cell types except megakaryocytes. Competing Interest Statement The authors have declared no competing interest.