Identification of a novel enhancer region 1.7 Mb downstream of the c-myc gene controlling its expression in hematopoietic stem and progenitor cells

The transcription factor and proto-oncogene c-Myc is a central regulator of cellular proliferation, growth, metabolism and differentiation in many cell types including stem cells. Although it is known that c-Myc expression is tightly controlled and can drive tumorigenesis if de-regulated, the mechanisms of its transcriptional regulation remain largely elusive. Besides its promoter, which is not sufficient to account for endogenous c-myc expression, only a few cis-regulatory elements have been defined. The c-myc gene is located on mouse chromosome 15 within a 4 Mb-long gene-poor region, which coincides with a large topologically associating domain (TAD). 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. Mutant HSCs were non-functional, as they lost multipotency and showed differentiation defects at the progenitor level, which resulted in their accumulation in vivo. This phenotype closely mimicked the phenotype of mice in which the c-myc gene was conditionally deleted by the Cre/loxP technique in the adult hematopoietic system using MxCre (MxCre; c-mycflox/flox). Importantly, gene expression analysis showed that the deletion of this enhancer region led to a dramatic reduction of c-myc expression in HSCs, multipotent progenitors and most mature cell types. Furthermore, compound heterozygous mice with one allele carrying the enhancer deletion and a c-myc null allele on the other chromosome displayed hematopoietic defects highly similar to MxCre; c-mycflox/flox animals, demonstrating genetic allelism between the c-myc coding region and the newly identified enhancer region. Altogether, these data provide genetic evidence that this enhancer region directly controls, in cis, c-myc expression in HSC/progenitor cells. Within the 126 kb enhancer region 8 modules of sizes between 0.4 kb and 1.9 kb were identified and analyzed for relative enrichment of the enhancer-associated H3K27ac histone mark by ChIP/qRT-PCR. This was performed in different hematopoietic lineages and revealed that distinct enhancer modules differentially control c-myc expression in granulocytes or HSC/progenitors. Interestingly, this evolutionary highly conserved enhancer region was shown to be focally amplified in a number of tumor samples from patients with acute myeloid leukemia (AML), suggesting that this enhancer may be a critical component driving increased c-MYC expression found in certain leukemias. In summary, we identified a very distant hematopoietic and stem/progenitor specific enhancer region for c-myc and provide genetic data supporting its critical function as a key regulatory region in normal hematopoiesis and a putative role in human AML

Identification of Regulatory Networks in HSCs and Their Immediate Progeny via Integrated Proteome, Transcriptome, and DNA Methylome Analysis

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

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 (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

Identification of DNA methylation changes at cis

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 DNA methylation changes at <i>cis</i>-regulatory elements during early steps of HSC differentiation using tagmentation-based whole genome bisulfite sequencing

<div><p>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 <i>cis</i>-acting regulatory regions that are dynamically remodeled during the first steps of haematopoietic differentiation.</p></div