Myc Depletion Induces a Pluripotent Dormant State Mimicking Diapause.

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.

Author Correction: A Myc enhancer cluster regulates normal and leukaemic haematopoietic stem cell hierarchies.

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).

A Myc enhancer cluster regulates normal and leukaemic haematopoietic stem cell hierarchies.

The transcription factor Myc is essential for the regulation of haematopoietic stem cells and progenitors and has a critical function in haematopoietic malignancies. Here we show that an evolutionarily conserved region located 1.7 megabases downstream of the Myc gene that has previously been labelled as a 'super-enhancer' is essential for the regulation of Myc expression levels in both normal haematopoietic and leukaemic stem cell hierarchies in mice and humans. Deletion of this region in mice leads to a complete loss of Myc expression in haematopoietic stem cells and progenitors. This caused an accumulation of differentiation-arrested multipotent progenitors and loss of myeloid and B cells, mimicking the phenotype caused by Mx1-Cre-mediated conditional deletion of the Myc gene in haematopoietic stem cells. This super-enhancer comprises multiple enhancer modules with selective activity that recruits a compendium of transcription factors, including GFI1b, RUNX1 and MYB. Analysis of mice carrying deletions of individual enhancer modules suggests that specific Myc expression levels throughout most of the haematopoietic hierarchy are controlled by the combinatorial and additive activity of individual enhancer modules, which collectively function as a 'blood enhancer cluster' (BENC). We show that BENC is also essential for the maintenance of MLL-AF9-driven leukaemia in mice. Furthermore, a BENC module, which controls Myc expression in mouse haematopoietic stem cells and progenitors, shows increased chromatin accessibility in human acute myeloid leukaemia stem cells compared to blasts. This difference correlates with MYC expression and patient outcome. We propose that clusters of enhancers, such as BENC, form highly combinatorial systems that allow precise control of gene expression across normal cellular hierarchies and which also can be hijacked in malignancies.

Hematopoietic stem cell quiescence and function are controlled by the CYLD-TRAF2-p38MAPK pathway.

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.

Transcriptome-wide profiling and posttranscriptional analysis of hematopoietic stem/progenitor cell differentiation toward myeloid commitment.

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 (Lin(neg)SCA-1(+)c-KIT(+)) and myeloid committed precursors (Lin(neg)SCA-1(neg)c-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.

Identification of DNA methylation changes at cis-regulatory elements during early steps of HSC differentiation using tagmentation-based whole genome bisulfite sequencing.

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.

Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.

In 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.