3D interactions with the growth hormone locus in cellular signalling and cancer-related pathways

Growth hormone (GH) is a peptide hormone predominantly produced by the anterior pituitary and is essential for normal growth and metabolism. The GH locus contains five evolutionarily related genes under the control of an upstream locus control region that coordinates tissue-specific expression of these genes. Compromised GH signalling and genetic variation in these genes has been implicated in various disorders including cancer. We hypothesised that regulatory regions within the GH locus coordinate expression of a gene network that extends the impact of the GH locus control region. We used the CoDeS3D algorithm to analyse 529 common single nucleotide polymorphisms (SNPs) across the GH locus. This algorithm identifies colocalised Hi-C and eQTL associations to determine which SNPs are associated with a change in gene expression at loci that physically interact within the nucleus. One hundred and eighty-one common SNPs were identified that interacted with 292 eGenes across 48 different tissues. One hundred and forty-five eGenes were regulated in trans. eGenes were found to be enriched in GH/GHR-related cellular signalling pathways including MAPK, PI3K-AKT-mTOR, ERBB and insulin signalling, suggesting that these pathways may be co-regulated with GH signalling. Enrichment was also observed in the Wnt and Hippo signalling pathways and in pathways associated with hepatocellular, colorectal, breast and non-small cell lung carcinoma. Thirty-three eQTL SNPs identified in our study were found to be of regulatory importance in a genome-wide Survey of Regulatory Elements reporter screen. Our data suggest that the GH locus functions as a complex regulatory region that coordinates expression of numerous genes in cis and trans, many of which may be involved in modulating GH function in normal and disease states.

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. Disclosures Melnick: Epizyme: Consultancy; Constellation: Consultancy; Janssen: Research Funding.

A Sir2-regulated locus control region in the recombination enhancer of Saccharomyces cerevisiae specifies chromosome III structure

The NAD+-dependent histone deacetylase Sir2 was originally identified in Saccharomyces cerevisiae as a silencing factor for HML and HMR, the heterochromatic cassettes utilized as donor templates during mating-type switching. MATa cells preferentially switch to MATα using HML as the donor, which is driven by an adjacent cis-acting element called the recombination enhancer (RE). In this study we demonstrate that Sir2 and the condensin complex are recruited to the RE exclusively in MATa cells, specifically to the promoter of a small gene within the right half of the RE known as RDT1. We go on to demonstrate that the RDT1 promoter functions as a locus control region (LCR) that regulates both transcription and long-range chromatin interactions. Sir2 represses the transcription of RDT1 until it is redistributed to a dsDNA break at the MAT locus induced by the HO endonuclease during mating-type switching. Condensin is also recruited to the RDT1 promoter and is displaced upon HO induction, but does not significantly repress RDT1 transcription. Instead condensin appears to promote mating-type switching efficiency and donor preference by maintaining proper chromosome III architecture, which is defined by the interaction of HML with the right arm of chromosome III, including MATa and HMR. Remarkably, eliminating Sir2 and condensin recruitment to the RDT1 promoter disrupts this structure and reveals an aberrant interaction between MATa and HMR, consistent with the partially defective donor preference for this mutant. Global condensin subunit depletion also impairs mating type switching efficiency and donor preference, suggesting that modulation of chromosome architecture plays a significant role in controlling mating type switching, thus providing a novel model for dissecting condensin function in vivo.Author summarySir2 is a highly conserved NAD+-dependent protein deacetylase and defining member of the sirtuin protein family. It was identified about 40 years ago in the budding yeast, Saccharomyces cerevisiae, as a gene required for silencing of the cryptic mating-type loci, HML and HMR. These heterochromatic cassettes are utilized as templates for mating-type switching, whereby a programmed DNA double-strand break at the MATa or MATα locus is repaired by gene conversion to the opposite mating type. The preference for switching to the opposite mating type is called donor preference, and in MATa cells, is driven by a cis-acting DNA element called the recombination enhancer (RE). It was believed that the only role for Sir2 in mating-type switching was silencing HML and HMR. However, in this study we show that Sir2 also regulates expression of a small gene (RDT1) in the RE that is activated during mating-type switching. The promoter of this gene is also bound by the condensin complex, and deleting this region of the RE drastically changes chromosome III structure and alters donor preference. The RE therefore appears to function as a complex locus control region (LCR) that links transcriptional control to chromatin architecture, and thus provides a new model for investigating the underlying mechanistic principles of programmed chromosome architectural dynamics.