A Myeloid-Specific Gene-Dosage Regulator for CEBPA Expression in Myeloid Cells Is Commonly Targeted By Onco-Proteins in AML
Authors: Avellino Roberto , Groschel Stefan M , Van de Werken Harmen , Sanders Mathijs A. , Hoogenboezem Remco M. , Erpelinck Claudia , Havermans Marije , Bindels Eric , Delwel Ruud 
Published: October 2019

Abstract
Changes in regulation of transcription factor (TF) networks are a hallmark of acute myeloid leukaemia (AML). Deregulation in expression of the leucine zipper transcription factor C/EBPa, is common in AML with recurrent oncogenic events, including 3q-rearrangementsEVI1+ or translocation t(8;21)RUNX1-ETO. We aimed to study how the chromatin organization of the CEBPA locus control transcription in myeloid cells and how AML-related oncoproteins functionally interfere with these enhancers to deregulate CEBPAexpression.
Human CEBPA is located on the short arm of chromosome 19 in a sub-topological associated domain that spans a genomic region of approximately 170kb. CEBPA is flanked by CEBPG and SLC7A10, at the 5’ and 3’, respectively. We hypothesized that upon myeloid commitment the 3D chromatin configuration of the locus facilitates the transcriptional activation of CEBPA by bringing enhancers in proximity to the CEBPA promoter and therefore enhances its expression. To identify potential enhancers that interact with the CEBPA promoter, we applied circularized chromatin conformation capture-sequencing (4C-seq) and compared the chromatin configuration of myeloidCEBPA+ (n=4) with that of lymphoidCEBPA- (n=2) cell lines. Using the CEBPA promoter as a viewpoint, all cell lines investigated showed a similar profile of interactions of multiple chromatin sites, localized within the locus. Non-haematopoietic cell lines also exhibited the same profile of interactions, suggesting no restricted chromatin configuration specific for haematopoietic origin. Our 4C-seq data show that the chromatin organization of the locus is highly conserved across different tissues, independent of CEBPA expression. However, the major differences between myeloidCEBPA+ cells and lymphoidCEBPA- are shown by the abundance of active enhancer marks, H3K27ac and p300 in chromatin immunoprecipitation-sequencing (ChIP-seq) analysis. These enhancers are located at the 3’ end of CEBPA and concentrated in a genomic cluster containing at least 6 potential enhancers. Non-haematopoieticCEBPA+ cells also showed multiple active enhance marks in ChIP-seq analysis at the 3’ end of CEBPA, but exhibited a different pattern than myeloidCEBPA+ cells, suggesting tissue specificity. At least two potential enhancers were exclusive for myeloid cells. These enhancers were investigated for transactivation potential in a luciferase based reporter system where only in myeloidCEBPA+ cells, as compared to non-haematopoieticCEBPA+cell lines, showed high transactivation.
Combining the complete 4C-seq and ChIP-seq set of data, we found a potential myeloid-specific enhancer as a strong candidate, located at +42kb downstream from CEBPA. A combination of in-house and public available ChIP-seq data show selective binding of haematopoietic stem cell and myeloid related TFs to this enhancer, such as LMO2, SCL, RUNX1, ERG, GATA2, and PU.1, in human CD34+ bone marrow cells and myeloid cell lines. In AML transformed cell lines and in human AML samples, EVI1 and AML1-ETO show strong binding to this enhancer by ChIP-seq, which may provide an explanation for the strongly reduced CEBPA expression levels in corresponding leukaemias. To study the role of this enhancer and CEBPA expression, we conducted CRISPR/Cas9 genome editing and deleted this enhancer (800bp) in a myeloidCEBPA+ cell line, THP-1. CEBPA expression was reduced in all targeted clones by 5 fold when compared to control clones.
In summary, we showed that the 3D chromatin organization of the human CEBPA locus is pre-configured at an early stage of development and thus, it is maintained across different cell types independent of CEBPA expression. Combining functional genomics data, we identified a novel human CEBPA enhancer important for CEBPA expression in myeloid cells. Oncogenic usage of this enhancer implies involvement in AML and can, partially, explain CEBPA deregulation in human AML subtypes.

Disclosures
No relevant conflicts of interest to declare.