An OCT2 / OCA-B / MEF2B Ternary Complex Controls the Activity and Architecture of an Essential Locus Control Region for Normal and Malignant Germinal Center B-Cells

The transcriptional repressor BCL6 is considered the master regulator of the germinal center (GC) reaction and is a key proto-oncogene in GC-derived lymphoma pathogenesis. At GC initiation, chromatin architecture is dramatically remodeled around a BCL6-associated locus control region (LCR) with presumed enhancer function. The BCL6 LCR is located 150kb upstream of the BCL6 gene and is fundamentally required for GC formation. Herein, we functionally dissect the BCL6 LCR to uncover the crucial genetic elements and transcription factor binding events that drive BCL6 LCR function.

We generated a custom gRNA library densely tiling the BCL6 LCR and surrounding regulatory elements. In total, the library covered a genomic region of 316.8kb with 25,698 gRNAs. We used a GC derived diffuse large B-cell lymphoma line, OCI-LY7, stably expressing dCas9-KRAB (CRISPRi) to screen for depletion of gRNAs. Surprisingly, BCL6 LCR function relied on only 4 of 21 constituent enhancers. These 4 essential enhancers were critically required for cell growth and BCL6 expression. In contrast, all other constituent enhancers were completely dispensable for LCR function. These results indicate that the BCL6 LCR is governed by a strong internal hierarchy and pinpoint the crucial genetic elements that drive LCR function.

To understand what distinguishes essential enhancers from non-essential enhancers, we interrogated ChIP-seq profiles of key GC transcription factors (TF) and transcriptional co-activators. While most TFs and co-activators bound constituent enhancers indiscriminately, MEF2B specifically bound only to essential enhancers (p=0.01). Interestingly, essential enhancers did not contain MEF2B binding motifs. Furthermore, MEF2B did not bind to essential enhancer DNA in electrophoretic mobility shift (EMSA) and immobilized template assays. De novo motif analysis of MEF2B ChIP-seq data prominently featured the canonical octamer transcription factor motif (p=10^-79) indicating a role for OCT2 in the recruitment of MEF2B to essential enhancers. Indeed, MEF2B binding to essential enhancer DNA in EMSA and immobilized template assays was dependent upon the presence of OCT2 and its co-activator OCA-B. We assayed occupancy of OCT2, OCA-B and MEF2B at essential enhancers in OCI-LY7 cells by qChIP and found that each factor required the other two for full binding activity. Furthermore, all three factors were required for BCL6 expression (p<0.001 for each factor). These results indicate that OCT2, OCA-B and MEF2B cooperatively bind to essential enhancer elements and act as an intimately linked ternary complex to drive BCL6 expression through the BCL6 LCR.

To elucidate how the OCT2 / OCA-B / MEF2B complex promotes target gene transcription, we performed IP of OCA-B followed by mass spectrometry and found highly significant interactions with the majority of Mediator proteins (p<10^-8). We furthermore showed that OCA-B directly and specifically interacts with MED1, through which it recruits the remainder of the Mediator complex and that OCA-B and OCT2 are required to recruit Mediator to essential enhancer elements.

The Mediator complex is thought to serve as a drawbridge across enhancers and promoters to facilitate enhancer-promoter looping. Using 3C assays, we found that OCA-B is required for chromatin contacts between the BCL6 promoter and the LCR highlighting its importance in recruiting Mediator. Similarly, essential enhancer elements were crucially required for intact chromatin conformation at the BCL6 locus as determined by 3C.

In summary, BCL6 LCR function completely relies on very few but highly essential enhancer elements. OCT2, OCA-B and MEF2B cooperatively bind these essential enhancers forming an intimately linked trimeric complex. By recruiting Mediator, OCA-B provides a direct link to the basal transcriptional machinery. Finally, essential enhancers as well as the OCT2 / OCA-B / MEF2B complex are required for BCL6 expression and intact chromatin conformation at the BCL6 locus - key determinants of the GC B cell state.