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In the originally published version of this Letter, ref. 43 was erroneously provided twice. In the 'Estimation of relative cell-type-specific composition of AML samples' section in the Methods, the citation to ref. 43 after the GEO dataset GSE24759 is correct. However, in the 'Mice' section of the Methods, the citation to ref. 43 after 'TAMERE' should have been associated with a new reference1. The original Letter has been corrected online (with the new reference included as ref. 49).

Mouse embryonic stem cells (ESCs) are maintained in a naive ground state of pluripotency in the presence of MEK and GSK3 inhibitors. Here, we show that ground-state ESCs express low Myc levels. Deletion of both c-myc and N-myc (dKO) or pharmacological inhibition of Myc activity strongly decreases transcription, splicing, and protein synthesis, leading to proliferation arrest. This process is reversible and occurs without affecting pluripotency, suggesting that Myc-depleted stem cells enter a state of dormancy similar to embryonic diapause. Indeed, c-Myc is depleted in diapaused blastocysts, and the differential expression signatures of dKO ESCs and diapaused epiblasts are remarkably similar. Following Myc inhibition, pre-implantation blastocysts enter biosynthetic dormancy but can progress through their normal developmental program after transfer into pseudo-pregnant recipients. Our study shows that Myc controls the biosynthetic machinery of stem cells without affecting their potency, thus regulating their entry and exit from the dormant state., This work was supported by the FOR2033 and SFB873 funded by the Deutsche Forschungsgemeinschaft (DFG), the Dietmar Hopp Foundation (all to A.T.), and the Wellcome Trust (to A.S.).

Tesio at al. identify a novel pathway controlled by the tumor suppressor and deubiquitinase cylindromatosis (CYLD), which is involved in the regulation of hematopoietic stem cell quiescence and repopulation potential., The status of long-term quiescence and dormancy guarantees the integrity of hematopoietic stem cells (HSCs) during adult homeostasis. However the molecular mechanisms regulating HSC dormancy remain poorly understood. Here we show that cylindromatosis (CYLD), a tumor suppressor gene and negative regulator of NF-κB signaling with deubiquitinase activity, is highly expressed in label-retaining dormant HSCs (dHSCs). Moreover, Cre-mediated conditional elimination of the catalytic domain of CYLD induced dHSCs to exit quiescence and abrogated their repopulation and self-renewal potential. This phenotype is dependent on the interactions between CYLD and its substrate TRAF2 (tumor necrosis factor–associated factor 2). HSCs expressing a mutant CYLD with an intact catalytic domain, but unable to bind TRAF2, showed the same HSC phenotype. Unexpectedly, the robust cycling of HSCs lacking functional CYLD–TRAF2 interactions was not elicited by increased NF-κB signaling, but instead by increased activation of the p38MAPK pathway. Pharmacological inhibition of p38MAPK rescued the phenotype of CYLD loss, identifying the CYLD–TRAF2–p38MAPK pathway as a novel important regulator of HSC function restricting HSC cycling and promoting dormancy.

Summary Hematopoietic stem cells possess lifelong self-renewal activity and generate multipotent progenitors that differentiate into lineage-committed and subsequently mature cells. We present a comparative transcriptome analysis of ex vivo isolated mouse multipotent hematopoietic stem/progenitor cells (LinnegSCA-1+c-KIT+) and myeloid committed precursors (LinnegSCA-1negc-KIT+). Our data display dynamic transcriptional networks and identify a stem/progenitor gene expression pattern that is characterized by cell adhesion and immune response components including kallikrein-related proteases. We identify 498 expressed lncRNAs, which are potential regulators of multipotency or lineage commitment. By integrating these transcriptome with our recently reported proteome data, we found evidence for posttranscriptional regulation of processes including metabolism and response to oxidative stress. Finally, our study identifies a high number of genes with transcript isoform regulation upon lineage commitment. This in-depth molecular analysis outlines the enormous complexity of expressed coding and noncoding RNAs and posttranscriptional regulation during the early differentiation steps of hematopoietic stem cells toward the myeloid lineage., Highlights • Transcriptional control and immune response pathways indicative of stem/progenitors • Identification of lncRNA species expressed during early adult hematopoiesis • Posttranscriptional regulation of oxidative stress response and metabolism • High degree of differential exon usage upon lineage commitment, In this article, Trumpp, Huber, and colleagues provide a comprehensive transcriptome and posttranscriptional analysis of hematopoietic stem/progenitor cell differentiation toward myeloid commitment. By integrating the proteome of these cells, they found evidence for posttranscriptional regulation in genes involved in metabolic stress response. They uncover lncRNA expression and transcript isoform changes upon commitment. All the data are available in an interactive website.

Epigenetic alterations during cellular differentiation are a key molecular mechanism which both instructs and reinforces the process of lineage commitment. Within the haematopoietic system, progressive changes in the DNA methylome of haematopoietic stem cells (HSCs) are essential for the effective production of mature blood cells. Inhibition or loss of function of the cellular DNA methylation machinery has been shown to lead to a severe perturbation in blood production and is also an important driver of malignant transformation. HSCs constitute a very rare cell population in the bone marrow, capable of life-long self-renewal and multi-lineage differentiation. The low abundance of HSCs has been a major technological barrier to the global analysis of the CpG methylation status within both HSCs and their immediate progeny, the multipotent progenitors (MPPs). Within this Extra View article, we review the current understanding of how the DNA methylome regulates normal and malignant hematopoiesis. We also discuss the current methodologies that are available for interrogating the DNA methylation status of HSCs and MPPs and describe a new data set that was generated using tagmentation-based whole genome bisulfite sequencing (TWGBS) in order to comprehensively map methylated cytosines using the limited amount of genomic DNA that can be harvested from rare cell populations. Extended analysis of this data set clearly demonstrates the added value of genome-wide sequencing of methylated cytosines and identifies novel important cis-acting regulatory regions that are dynamically remodeled during the first steps of haematopoietic differentiation.

In the originally published version of this Letter, ref. 43 was erroneously provided twice. In the 'Estimation of relative cell-type-specific composition of AML samples' section in the Methods, the citation to ref. 43 after the GEO dataset GSE24759 is correct. However, in the 'Mice' section of the Methods, the citation to ref. 43 after 'TAMERE' should have been associated with a new reference1. The original Letter has been corrected online (with the new reference included as ref. 49).

SummaryIn this study, we present integrated quantitative proteome, transcriptome, and methylome analyses of hematopoietic stem cells (HSCs) and four multipotent progenitor (MPP) populations. From the characterization of more than 6,000 proteins, 27,000 transcripts, and 15,000 differentially methylated regions (DMRs), we identified coordinated changes associated with early differentiation steps. DMRs show continuous gain or loss of methylation during differentiation, and the overall change in DNA methylation correlates inversely with gene expression at key loci. Our data reveal the differential expression landscape of 493 transcription factors and 682 lncRNAs and highlight specific expression clusters operating in HSCs. We also found an unexpectedly dynamic pattern of transcript isoform regulation, suggesting a critical regulatory role during HSC differentiation, and a cell cycle/DNA repair signature associated with multipotency in MPP2 cells. This study provides a comprehensive genome-wide resource for the functional exploration of molecular, cellular, and epigenetic regulation at the top of the hematopoietic hierarchy.

The c-MYC transcription factor is a central regulator of cellular proliferation, growth, metabolism and differentiation in many cell types including stem cells (1). Although it is known that c-Myc expression is tightly controlled and can drive transformation if de-regulated, the mechanisms of its transcriptional regulation remain elusive. Besides its promoter, which is not sufficient to account for Myc endogenous expression, only a few cis-regulatory elements have been defined. c-Myc is located within a 4 Mb-long gene-poor region, which coincides with a large topologically associating domain (TAD) (2). At the distal end of this TAD, 1.7 Mb downstream of the mouse c-Myc gene, we identified a cluster of enhancer-associated chromatin marks which were present only in hematopoietic tissues. A LacZ reporter gene inserted next to this cluster showed specific expression in hematopoietic stem cells (HSCs) and progenitor cells. Mice homozygous for a deletion of this enhancer region showed decreased myeloid and B cells, while HSCs, multipotent progenitors and megakaryocytes accumulated in the bone marrow. This phenotype closely mimicked the phenotype of mice in which the c-Myc gene was conditionally deleted using Mx1-Cre (3). Importantly, gene expression analysis showed that the deletion of this enhancer region led to a dramatic reduction of Myc expression in HSCs, multipotent progenitors and most mature cell types. Furthermore, compound heterozygous mice carrying one enhancer deletion allele and one c-Myc null allele demonstrated very similar hematopoietic defects to that of homozygous c-Myc null mice, showing allelism between c-Myc and the enhancer region. Altogether, these data provide genetic evidence that this enhancer region directly controls, in cis, c-Myc expression in HSC/progenitor cells. As the region is composed of multiple modules, we examined the relative enrichment of the enhancer-associated H3K27ac mark by ChIP for the different modules in different hematopoietic lineages. This analysis suggested that selective enhancer elements contribute differently to c-Myc expression in either granulocytes or HSC/progenitors. Interestingly, this highly conserved enhancer region is focally amplified in a number of AML patients, suggesting that it may be a critical component driving the increased Myc expression found in leukemias. In summary, we identified a distant hematopoietic-specific enhancer region for c-Myc and provide data which supports its critical function as a key regulatory region in normal hematopoiesis and highlights a potential role in leukemic transformation. (1) Laurenti et al. (2008). Hematopoietic stem cell function and survival depend on c-Myc and N-Myc activity. Cell Stem Cell 3(6): 611-624. (2) Dixon et al. (2012). Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485(7398): 376-380. (3) Wilson et al. (2004). c-Myc controls the balance between hematopoietic stem cell self-renewal and differentiation. Genes Dev 18(22): 2747-2763. Disclosures No relevant conflicts of interest to declare.