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