Abstract 4993: The role of super-enhancers and genetic boundaries in gene regulation

The purpose of this study was to investigate the role of the Zn finger protein CTCF in the establishment of genetic borders and in controlling spatially-restricted activity of a mammary-specific super-enhancer (SE) known to selectively activate gene expression during pregnancy. CRISPR/Cas9 gene editing was used to generate mice with deletions in CTCF binding sites (CBSs) separating the Wap SE from the Ramp3 gene. ChIP-seq and qRT-PCR were used to investigate protein-DNA interactions and changes in gene expression, respectively. RNA-seq data demonstrated that expression of the mammary-specific Wap and widely-expressed Ramp3 gene was activated in mammary tissue at the onset of lactation. To investigate the possibility that the Wap SE can regulate both genes, mice carrying deletions in the SE were established. In these mice, both Wap and downstream Ramp3 were downregulated to the same extent, supporting the notion that the SE regulates both genes. Given these results, we investigated the contributions of several intervening CBSs in dampening SE activity on Ramp3. Mice harboring individual and combinatorial deletions of three CBSs (A, B and C) between the Wap SE and Ramp3 were generated. While loss of CTCF at site B (ΔB) had no apparent effect, loss of site A (ΔA) led to an expansion of H3K27ac from the adjacent SE in mammary tissue. The site C mutation (ΔC) resulted in a two-fold induction of Ramp3 in mammary tissue. Mice lacking all three binding sites demonstrated a marked increase in Ramp3 expression, which indicates that the CBSs block expression in an additive manner. To test the possible insulator function of these in a tissue with opposing gene expression, cerebellum was chosen as Ramp3 is actively transcribed while Wap is silent. The ΔA and ΔB mice showed no phenotype, but surprisingly ΔC mice demonstrated an 80% reduction in Ramp3 expression. Analysis of ChIP-seq and DNAse-seq demonstrated that site C coincides with a cerebellum-specific enhancer, which appears to regulate Ramp3 expression. These data demonstrate that a SE can activate two genes, albeit to different degrees. Furthermore, we demonstrate that CBSs do not serve as tight genetic boundaries encapsulating transcriptional enhancers and their associated genes, but are porous borders that allow for fine tuning of transcriptional activity. Individual CBSs have a modest ability to insulate genes, but a cluster of CTCF proteins is required to adjust the enhancer activity on genes and prevent deregulation. In addition, we show that CBSs are not restricted to a unique function and can act in a tissue-specific manner to contribute to gene regulation. These data help us to develop a more nuanced understanding of SE function as well as the roles of CTCF in regulating gene expression. Previous studies have shown that SEs and CBSs are mutational hotspots in cancer, implicating them in tumorigenesis. A more complete understanding of their biology will allow a better understanding of their role in cancer. Citation Format: Michaela Willi, Tyler M. Kuhns, Kyung Hyun Yoo, Fabian Reinisch, Christian Reinbold, Teresa Mohr, Chaochen Wang, Lothar Hennighausen. The role of super-enhancers and genetic boundaries in gene regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4993. doi:10.1158/1538-7445.AM2017-4993