A Novel Enhancer Region 1.7Mb Downstream of the C-Myc Gene Drives Its Expression in Hematopoietic Stem and Progenitor Cells

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.