HoxA9 binds and represses the Cebpa +8 kb enhancer

Abstract

C/EBPα plays a key role in specifying myeloid lineage development. HoxA9 is expressed in myeloid progenitors, with its level diminishing during myeloid maturation, and HOXA9 is over-expressed in a majority of acute myeloid leukemia cases, including those expressing NUP98-HOXD13. The objective of this study was to determine whether HoxA9 directly represses Cebpa gene expression. We find 4-fold increased HoxA9 and 5-fold reduced Cebpa in marrow common myeloid and LSK progenitors from Vav -NUP98-HOXD13 transgenic mice. Conversely, HoxA9 decreases 5-fold while Cebpa increases during granulocytic differentiation of 32Dcl3 myeloid cells. Activation of exogenous HoxA9-ER in 32Dcl3 cells reduces Cebpa mRNA even in the presence of cycloheximide, suggesting direct repression. Cebpa transcription in murine myeloid cells is regulated by a hematopoietic-specific +37 kb enhancer and by a more widely active +8 kb enhancer. ChIP-Seq analysis of primary myeloid progenitor cells expressing exogenous HoxA9 or HoxA9-ER demonstrates that HoxA9 localizes to both the +8 kb and +37 kb Cebpa enhancers. Gel shift analysis demonstrates HoxA9 binding to three consensus sites in the +8 kb enhancer, but no affinity for the single near-consensus site present in the +37 kb enhancer. Activity of a Cebpa +8 kb enhancer/promoter-luciferase reporter in 32Dcl3 or MOLM14 myeloid cells is increased ~2-fold by mutation of its three HOXA9-binding sites, suggesting that endogenous HoxA9 represses +8 kb Cebpa enhancer activity. In contrast, mutation of five C/EBPα-binding sites in the +8 kb enhancer reduces activity 3-fold. Finally, expression of a +37 kb enhancer/promoter-hCD4 transgene reporter is reduced ~2-fold in marrow common myeloid progenitors when the Vav- NUP98-HOXD13 transgene is introduced. Overall, these data support the conclusion that HoxA9 represses Cebpa expression, at least in part via inhibition of its +8 kb enhancer, potentially allowing normal myeloid progenitors to maintain immaturity and contributing to the pathogenesis of acute myeloid leukemia associated with increased HOXA9.

Integrative genomic and transcriptomic profiling reveals distinct molecular subsets in adult mixed phenotype acute leukemia

“…According to the literature, Hoxa9 overexpression in mice leads to the occurrence of precursor T‐cell lymphoblastic leukemia/lymphoma with NOTCH1 mutations 29 . In turn, Hoxa9 was reported to repress Cebpa expression, at least in part, by inhibiting its enhancer 30 . Hence, all the above findings indicate that HOXA9 overexpression might play a key role in the mutual exclusivity of NOTCH1 and CEBPA .…”

“…Cebpa expression, at least in part, by inhibiting its enhancer. 30 36,37 RUNX1-mutated AML also exhibited a distinct upregulation of genes involved in B-cell development. 38 Therefore, the RUNX1 mutation could be an early event in the onset of Ph+ MPAL.…”

Deregulation of Transcriptional Enhancers in Cancer

“…Overexpressing HMGN1 in progenitor cells impairs their differentiation, promotes their proliferation, and causes a global increase in chromatin accessibility. The overexpression of HMGN1 therefore leads to the upregulation of oncogenes and loss of lineage-specifying regulators, such as C/EBPα, resulting in an expression profile similar to that of leukaemia stem cells [ 78 , 79 , 80 ]. The contribution of deregulated enhancers to neoplastic transformation is more evident in aging HSCs that accumulate mutations in epigenetic regulators.…”

The contributions of DNA accessibility and transcription factor occupancy to enhancer activity during cellular differentiation

“…The enhancer located at 37kb is bound by PU.1, other Ets TFs, C/EBP, Runx1, SCL, Gata2, and Myb [17, 18, 30, 31] in myeloid cells. The enhancer at 8kb is bound by C/EBP and Gfi1 [18, 31, 32] and the third enhancer is bound by PU.1 [18, 31]. While the binding sites and identities of the TFs regulating Cebpa enhancers have been established, it is not understood how the regulatory contributions of these TFs modulate Cebpa ’s gene expression during myeloid differentiation.…”