Two Hematopoietic Transcription Factors, RUNX1 and FUBP1, Control the Expression of KIT Oncogene in Pre-B Lymphoblasts

Hematopoiesis involves an elaborate network of transcription factors articulated with transcriptional cooperators. RUNX1 geneencodes a transcription factor playing a key role in hematopoiesis. Abnormal functions of RUNX1 protein are implicated in blood cancer, notably in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). To unravel the mechanisms behind the modulation of the transcriptional activity of RUNX1, we performed experiments of RUNX1-specific chromatin immunoprecipitation (ChIP) followed by mass spectrometry, and identified Far Upstream Element Binding Protein 1 (FUBP1) as a potential functional cooperator of RUNX1. The helicase and transcriptional regulator FUBP1 has recently been described to be essential for expansion and self-renewal of hematopoietic stem cells and to function as a potential cancer driver gene in lymphoblastic leukemia. Our goals were to validate the protein interaction between RUNX1 and its putative cooperator FUBP1, to identify gene(s) transcriptionally controlled by both RUNX1 and FUBP1, and finally to demonstrate the biological consequence of the cooperation between RUNX1 and its cooperator FUBP1.

Co-immunoprecipitation and colocalization experiments demonstrate that RUNX1 colocalizes with FUBP1 in pre-B cell lines, in pre-B lymphoblasts isolated from BCP-ALL patients, and more generally in other hematopoietic progenitors. To characterize some target genes directly regulated by FUBP1 and RUNX1, we performed ChIP combined with sequencing (ChIP-seq) for FUBP1 and RUNX1. Those binding signals were compared to different histone marks and reveal that a subset of FUBP1 and RUNX1 binding signals are enriched within primed enhancer regions characterized by histone H3K4me1 marks. Our data show that RUNX1 signal is also enriched within FUBP1 regions, demonstrating that they are often retrieved on same chromatin regions, bound by FUBP1. Interestingly, FUBP1 is widely distributed in the genome, therefore only a subset of RUNX1 binding signals overlaps with FUBP1 binding signals. To go further, using qPCR, we investigated the expression level of several potential target candidate genes in pre-B cells overexpressed for FUBP1 or RUNX1. We found that both FUBP1 and RUNX1 positively regulate KIT transcript. KIT is a tyrosine kinase receptor expressed on hematopoietic progenitors cell surface and is an oncogene overexpressed notably in different types of leukemia. With the comparison of data from FUBP1 and RUNX1 ChiP-seq, we found that FUBP1 and RUNX1 share two common DNA binding regions on KIT gene, including one overlapping with histone H3K4me1 mark. ChIP-qPCR analyses confirm the binding of FUBP1 and RUNX1 on the KIT enhancer. Moreover, using reporter gene assay, we demonstrated that they can both activate this enhancer. With supervised and unsupervised approaches, we were able to determine FUBP1 and RUNX1 binding motifs that are functionally important for the enhancer activation. Finally, we demonstrated that FUBP1 overexpression in a pre-B cell line increases the expression of KIT protein and exacerbates one of the KIT downstream pathways. FUBP1 overexpression increases also cell proliferation by promoting cell cycle progression in vitro . Concordantly, in vivo, xenografted immunodeficient mice injected with overexpressed FUBP1 cells present a shorter survival time than control mice.

To conclude, we demonstrate a regulatory network involving RUNX1 and FUBP1 for the control of a KIT enhancer. Indeed, FUBP1 and RUNX1 can colocalize on chromatin, regulate KIT transcription by binding to a common region in a KIT enhancer, and promote cell cycle progression. These findings suggest a new mechanism for the control of the proliferation of pre-B lymphoblasts. Because FUBP1 and KIT are overexpressed in some types of leukemia, abnormalities in this regulatory network could participate in the onset or the maintenance of RUNX1-related leukemia.

This work is supported by Ligue Régionale Contre le Cancer, Région Bretagne, Rennes Métropole, French Research Ministry, Société Française de lutte contre les Cancers et les leucémies de l'Enfant et de l'adolescent, Fédération Enfants et Santé, Société Française de Biochimie et Biologie Moléculaire, Société Française d'Hématologie, Mrs. M-Dominique Blanc-Bert and the Marie Curie Actions of the European Union FP7 under REA grant agreement n°291851.