HoxA9 binds and represses the Cebpa +8 kb enhancer

Lei Peng, Hong Guo, Peilin Ma, Yuqing Sun, Lauren Dennison, Peter D. Aplan, Jay Hess, Alan D. Friedman

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article numbere0217604
JournalPLoS ONE
Volume14
Issue number5
DOIs
StatePublished - May 1 2019
Externally publishedYes

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Myeloid Progenitor Cells
Myeloid Cells
Transgenes
Acute Myeloid Leukemia
myeloid leukemia
Bone Marrow
Binding Sites
transgenes
binding sites
Mutation
promoter regions
Electrophoretic Mobility Shift Assay
cells
Transcription
Cycloheximide
Luciferases
mutation
Gene expression
Transgenic Mice
mice

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Peng, L., Guo, H., Ma, P., Sun, Y., Dennison, L., Aplan, P. D., ... Friedman, A. D. (2019). HoxA9 binds and represses the Cebpa +8 kb enhancer. PLoS ONE, 14(5), [e0217604]. https://doi.org/10.1371/journal.pone.0217604

HoxA9 binds and represses the Cebpa +8 kb enhancer. / Peng, Lei; Guo, Hong; Ma, Peilin; Sun, Yuqing; Dennison, Lauren; Aplan, Peter D.; Hess, Jay; Friedman, Alan D.

In: PLoS ONE, Vol. 14, No. 5, e0217604, 01.05.2019.

Research output: Contribution to journalArticle

Peng, L, Guo, H, Ma, P, Sun, Y, Dennison, L, Aplan, PD, Hess, J & Friedman, AD 2019, 'HoxA9 binds and represses the Cebpa +8 kb enhancer', PLoS ONE, vol. 14, no. 5, e0217604. https://doi.org/10.1371/journal.pone.0217604
Peng L, Guo H, Ma P, Sun Y, Dennison L, Aplan PD et al. HoxA9 binds and represses the Cebpa +8 kb enhancer. PLoS ONE. 2019 May 1;14(5). e0217604. https://doi.org/10.1371/journal.pone.0217604
Peng, Lei ; Guo, Hong ; Ma, Peilin ; Sun, Yuqing ; Dennison, Lauren ; Aplan, Peter D. ; Hess, Jay ; Friedman, Alan D. / HoxA9 binds and represses the Cebpa +8 kb enhancer. In: PLoS ONE. 2019 ; Vol. 14, No. 5.
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