Normal RUNX1 and Pathogenic ETV6/RUNX1 Compete Genome-Wide for Chromatin Binding in Pre-B Acute Lymphoblastic Leukemia

Introduction and objectives:

The ETV6/RUNX1 fusion protein is a leukemia-initiating transcription factor that interferes with normal RUNX1 transcription factor function. Although RUNX1 can be either a repressor or an activator, ETV6/RUNX1 is mainly associated with transcriptional repression. Both RUNX1 and ETV6/RUNX1 proteins are expressed in pre-B acute lymphoblasts and bind the same DNA binding motif, which is normally recognized by the RUNX1 DNA binding domain, therefore competing for gene regulation with possible adverse transcriptional effects. Here, (we address the question of how) do the abnormal transcription factor ETV6/RUNX1 and the normal RUNX1 interplay at the chromatin level to modulate expression of genes, possibly giving rise to leukemia.

Materials and methods:

We used chromatin-immunoprecipitation coupled with sequencing (ChIP-seq) both in pre-B leukemic REH cells, which originate from a relapse of an ETV6/RUNX1 leukemic patient and lack the non-translocated allele of ETV6, and in pre-B leukemic Nalm6 cells that are ETV6/RUNX1-negative, to characterize the genomic distribution of ETV6/RUNX1 and RUNX1, as well as transcriptionnally active and inactive chromatin regions (ChIP-seq for methylated H3K4me3 and H3K4me1 histones, and acetylated H3K27achistones). In addition, we ran microarrays experiments to define the transcriptional programme associated with ETV6/RUNX1 and RUNX1 expression in pre-B cell lines. Data were compared to gene expression data from ETV6/RUNX1 pre-B acute lymphoblastic leukemia samples.

Results:

Through mapping the genome-wide distribution of RUNX1 et ETV6/RUNX1, we identified both common and exclusive DNA sites. We further characterized the DNA binding motifs of RUNX1 and ETV6/RUNX1, which were particularly enriched within particular transposon sequences. ChIP-seq of histone marks identified active and inactive chromatin in correlation with ETV6/RUNX1- and RUNX1-binding. Direct target genes for both transcription factors were defined and used for gene ontology analysis, allowing to uncover specific biological functions associated with ETV6/RUNX1 and RUNX1 binding events. Finally, we show that a fraction of the genes misregulated in pre-B acute lymphoblastic leukemia patients are directly controlled by ETV6/RUNX1 and RUNX1.

Conclusion:

Our data give new insights on the competition between the fusion protein ETV6/RUNX1 and normal RUNX1 in pre-B acute lymphoblastic leukemia at chromatin level genome-wide.

This work was supported by CNRS, University of Rennes 1, University Hospital of Rennes, Ligue Régionale contre le Cancer (sections 22, 35 and 56) (MPA, VG, MBT), Région Bretagne (MBT, VG), Rennes Métropole (MBT), SFR Biosit UMS 3480 (VG, MBT), Société Française de Lutte contre les Cancers et les Leucémies de l’Enfant et de l’Adolescent and the Fédération Enfants et Santé (MBT), Association Laurette Fugain (VG) and the People Programme (Marie Curie Actions) of the European Union Seventh Framework Programme (FP7/2007-2013) under REA grant agreement n°291851 (MBT).