Altered Enhancer Activity Represses CEBPE Expression in MLL -Rearranged Leukemia

Abstract

Mixed lineage leukemia (MLL)-rearranged leukemia is characterized by the presence of MLL fusion proteins resulting from chromosomal translocation between MLL and its partner genes. The resultant fusion proteins can confer aberrant epigenetic patterns at gene regulatory elements to disrupt transcriptional program, and ultimately leads to differentiation blockage at early hematopoietic progenitor stage. CCAAT enhancer binding proteins (C/EBPs) are key transcription factors involved in myeloid lineage commitment and maturation, among which CEBPE is crucial in terminal differentiation of granulocytes/macrophages. We found that Cebpe is markedly downregulated in murine hematopoietic stem/progenitor cells (HSPCs) expressing MLL-EEN, which could be associated with differentiation blockage in MLL -rearranged leukemia. However, how CEBPE is epigenetically dysregulated by MLL fusion proteins remains unclear. In murine granulocytes, we demonstrated long-range chromatin interaction between Cebpe promoter and enhancer by chromatin conformation capture (3C) assay, conferring the strong expression of Cebpe . Strikingly, we noticed gain of DNA methylation at Cebpe promoter in MLL-EEN expressing HSPCs, suggesting the disruption of chromatin interaction through aberrant epigenetic alterations resulted in Cebpe repression. Interestingly, CEBPE expression was also found significantly lower in human MLL acute myeloid leukemia (AML) cell lines (HL60, NB4, U937 and Kasumi-1), when compared to non-MLL AML cell lines (ML-2, MV4:11, Monomac-6 and THP-1). DNA hypermethylation was observed at both promoter and predicated enhancer regions in MLL AML cells, which were absent in non-MLL AML cells. In addition, reduction of H3K27ac enrichment at the predicted enhancer was found in MLL AML cells. Analysis of the public available RNA Polymerase II (RNAPII) ChIP-seq data demonstrated RNAPII binding at both CEBPE promoter and enhancers in non-MLL AML cell lines, but it was absent at the enhancer region in MLL AML cells. These results suggest a common epigenetic mechanism which disrupts the CEBPE enhancer-promoter interaction in MLL -rearranged leukemia. Besides, treatment of MLL AML cell lines with DNA methylation inhibitor 5'-aza-cytidine (5-aza-C) demonstrated induction of CEBPE and myeloid marker CD11b . While all -trans retinoid acid failed to trigger cell differentiation in ML-2 AML cells, co-treatment with 5-aza-C resulted in further induction of CEBPE and CD11b, compared to 5-aza-C treatment per se . Collectively, our work shows that aberrant DNA methylation mediated by MLL fusion proteins represses CEBPE expression through altering its enhancer activity, which subsequently leads to cell differentiation blockage in MLL AML cells. This study provides novel insights on the aberrant epigenetic regulation in MLL -rearranged leukemia and facilitates the development of differentiation therapy for leukemia.