The +37 kb Cebpa Enhancer Is Critical for Cebpa Myeloid Gene Expression and Contains Functional Sites that Bind SCL, GATA2, C/EBPα, PU.1, and Additional Ets Factors.

Stacy Cooper,Hong Guo,Alan D Friedman,Thomas Langmann

doi:10.1371/journal.pone.0126385

Abstract

The murine Cebpa gene contains an evolutionarily conserved 453 bp enhancer located at +37 kb that, together with its promoter, directs expression to myeloid progenitors and to long-term hematopoietic stem cells in transgenic mice. In human acute myeloid leukemia cases, the enhancer lacks point mutations but binds the RUNX1-ETO oncoprotein. The enhancer contains the H3K4me1 and H3K27Ac histone modifications, denoting an active enhancer, at progressively increasing levels as long-term hematopoietic stem cells transition to granulocyte-monocyte progenitors. We previously identified four enhancer sites that bind RUNX1 and demonstrated that their integrity is required for maximal enhancer activity in 32Dcl3 myeloid cells. The +37 kb Cebpa enhancer also contains C/EBP, Ets factor, Myb, GATA, and E-box consensus sites conserved in the human +42 kb CEBPA enhancer. Mutation of the two C/EBP, seven Ets, one Myb, two GATA, or two E-box sites reduces activity of an enhancer-promoter reporter in 32Dcl3 cells. In 293T gel shift assays, exogenous C/EBPα binds both C/EBP sites, c-Myb binds the Myb site, PU.1 binds the second Ets site, PU.1, Fli-1, ERG, and Ets1 bind the sixth Ets site, GATA2 binds both GATA sites, and SCL binds the second E-box. Endogenous hematopoietic RUNX1, PU.1, Fli-1, ERG, C/EBPα, GATA2, and SCL were previously shown to bind the enhancer, and we find that endogenous PU.1 binds the second Ets site in 32Dcl3 cells. Using CRISPR/Cas9, we developed 32Dcl3 lines in which the wild-type enhancer alleles are replaced with a variant mutant in the seven Ets sites. These lines have 20-fold reduced Cebpa mRNA when cultured in IL-3 or G-CSF, demonstrating a critical requirement for enhancer integrity for optimal Cebpa expression. In addition, these results indicate that the +37 kb Cebpa enhancer is the focus of multiple regulatory transcriptional pathways that impact its expression during normal hematopoiesis and potentially during myeloid transformation.

Highlights

CCAAT/enhancer binding protein α (C/EBPα) is a basic region-leucine zipper transcription factor expressed preferentially within granulocytic and monocytic myeloid cells during hematopoiesis [1]
We demonstrate that mutation of conserved C/EBP, Ets, Myb, GATA, and E-box sites each reduce enhancer activity in 32Dcl3 cells and utilize gel shift assays to show that C/EBPα, PU.1, Fli-1, ERG, Ets1, c-Myb, GATA2, and SCL have the capacity to bind one or more of their consensus sites within the enhancer and that PU.1 is the most prominent Ets factor that binds the enhancer in 32Dcl3 cells
Cells were split between media containing IL-3 or G-CSF, the latter known to induce endogenous Cebpa transcription and granulocytic differentiation [1], and cell extracts were analyzed for luciferase and β-galactosidase activities 42 hr later (Fig 2A)

Summary

Introduction

CCAAT/enhancer binding protein α (C/EBPα) is a basic region-leucine zipper transcription factor expressed preferentially within granulocytic and monocytic myeloid cells during hematopoiesis [1]. The Cebpa promoter is auto-activated by C/EBPα and contains two near-consensus RUNX1 cis elements that bind RUNX1 in gel shift assay and chromatin immunoprecipitation (ChIP) assays and are functional in the 32Dcl myeloid cell line [10,11]. The Cebpa enhancer binds endogenous RUNX1 as assessed by ChIP and contains four perfect RUNX1 consensus sites that bind RUNX1 in gel shift assay; mutation of these sites obviated 6-fold enhancer induction of promoter activity in 32Dcl cells [11]. RUNX1, as well as C/EBPα, PU., ERG, Fli-1, GATA2, SCL, Meis, and Gfi-1b, had been found to bind chromatin in the region of this enhancer in hematopoietic cells, as determined by ChIP-Seq [13,14]

Methods

Results

Conclusion