CRISPRi screen identifies core regions in IGH enhancers essential for non-Hodgkin lymphoma cells survival

Chromosomal translocations in non-Hodgkin lymphoma (NHL) are known to bring oncogenes (e.g. MYC, BCL2) under the regulation of immunoglobulin heavy chain (IGH) super-enhancers, composed of the intronic Eµ enhancer and 3’ regulatory regions (3’RR1, 3’RR2). Deregulation of translocated oncogenes by enhancer activity can contribute to lymphomagenesis. The role of IGH enhancers in normal B-cell development is well established, but the knowledge regarding precise mechanisms of their involvement in control of the translocated oncogenes is limited. We previously identified exact regions within IGH regulatory elements, one in the Eµ and two in each of the 3'RRs, whose targeting profoundly inhibited NHL cells growth. The goal of this project was to reveal enhancer RNAs (eRNA) transcribed from Eµ and 3'RRs and their essential regions. With the use of chromatin-enriched RNA-Seq we confirmed bi-directional transcription from those core enhancer regions and further validated eRNAs expression in a large panel of NHL cell lines and in patient-derived samples. We showed in several NHL cell lines that CRISPR/dCas9-KRAB-mediated inhibition of essential IGH enhancer regions decreased expression of eRNAs and translocated oncogenes in the fashion likely related to their breakpoints pattern within IGH locus. Our results provide new insights into the current understanding of IGH enhancers role in B-cell NHLs.