Identification and characterization of E2A-PBX1 genomic binding sites using ChIP-seq and bioinformatics
Bioinformatics , Cancer Biology , Epigenetics , Next-Generation Sequencing , Leukemia , Transcription Factors
A translocation between chromosomes 1 and 19, referred to as t(1;19), results in expression of the chimeric, oncogenic transcription factor E2A-PBX1 in cases of B-progenitor acute lymphoblastic leukemia. Previous studies have shown that E2A-PBX1 functions as an activator of target gene transcription and that oncogenesis requires direct binding of E2A-PBX1 with the histone acetyltransferase CBP/p300. However, the transcriptional targets of this complex remain largely unknown. To identify and characterize genomic sites bound by E2A-PBX1 we performed RNA-seq and ChIP-seq for E2A-PBX1, p300 and the functional histone marks H3K4me1, H3K4me3, and H3K27me3 in the t(1;19) leukemic cell line RCH-ACV. Putative E2A-PBX1 binding sites were validated by site-specific ChIP-qPCR. Bioinformatics tools were used to perform “integrative analysis” and characterize E2A-PBX1 binding on a genome-wide scale. This analysis resulted in the identification of 555 high-confidence E2A-PBX1 sites in the RCH-ACV genome most of which occur distally (>10kb) relative to the TSSs of known genes. Ontology and pathway analysis showed that candidate E2A-PBX1 target genes are involved in hematopoiesis, lymphopoiesis, and cancer progression. Binding of E2A-PBX1 to a random sampling of these sites was performed using site-specific ChIP-qPCR. Consistent with earlier results, E2A-PBX1-bound sites were strongly associated with p300 recruitment and the activating chromatin marks H3K4me1 and H3K4me3; analysis of RNA-seq data confirmed a strong association between proximate binding by E2A-PBX1 and gene transcription. Surprisingly, rather than associating with consensus PBX1 binding sequences as expected, E2A-PBX1 associated with sites bound by the B-lymphopoietic transcription factors EBF1 and wild-type E2A; EBF1, E2A and E2A-PBX1 were confirmed to co-localize at selected E2A-PBX1 binding sites. The results of this study identify hundreds of E2A-PBX1 genomic binding sites, confirm CBP/p300 recruitment and transcriptional activation in association with these sites, and raise the novel and surprising possibility that E2A-PBX1 disrupts the transcriptional regulation of B-lymphopoiesis through physically associating with the lymphopoietic transcription factors EBF1 and wild-type E2A.