Functional analysis of limb transcriptional enhancers in the mouse

Abstract

Transcriptional enhancers are genomic sequences bound by transcription factors that act together with basal transcriptional machinery to regulate gene transcription. Several high-throughput methods have generated large datasets of tissue-specific enhancer sequences with putative roles in developmental processes. However, few enhancers have been deleted from the genome to determine their roles in development. To understand the roles of two enhancers active in the mouse embryonic limb bud we deleted them from the genome. Although the genes regulated by these enhancers are unknown, they were selected because they were identified in a screen for putative limb bud-specific enhancers associated with p300, an acetyltransferase that participates in protein complexes that promote active transcription, and because the orthologous human enhancers (H1442 and H280) drive distinct lacZ expression patterns in limb buds of embryonic day (E) 11.5 transgenic mice. We show that the orthologous mouse sequences, M280 and M1442, regulate dynamic expression in the developing limb. Although significant transcriptional differences in enhancer-proximal genes in embryonic limb buds accompany the deletion of M280 and M1442 no gross limb malformations during embryonic development were observed, demonstrating that M280 and M1442 are not required for mouse limb development. However, M280 is required for the development and/or maintenance of body size; M280 mice are significantly smaller than controls. M280 also harbors an “ultraconserved” sequence that is identical between human, rat, and mouse. This is the first report of a phenotype resulting from the deletion of an ultraconserved element. These studies highlight the importance of determining enhancer regulatory function by experiments that manipulate them in situ and suggest that some of an enhancer's regulatory capacities may be developmentally tolerated rather than developmentally required.

Genetic Variations of Ultraconserved Elements in the Human Genome

“…(Table 1). Our results imply that conservation of at least some UCEs still is of high importance for normal phenotype, which is in accordance with published UCEs knockout studies (Dickel et al, 2018;Nolte et al, 2014).…”

“…Initial deletion studies suggested that the absence of UCEs did not result in overt phenotypic changes in mice (Ahituv et al, 2007). More recently, however, clear phenotypic effects were detected when other UCEs were deleted from the mouse genome (Dickel et al, 2018;Nolte et al, 2014). A large fraction of UCEs have been found to be transcriptionally active and involved in multiple human cancers (Calin et al, 2007;reviewed in Fabris and Calin, 2017;Terracciano et al, 2017).…”

Loss-of-function tolerance of enhancers in the human genome

“…However, further follow-up studies found that even though deletion of ultra-conserved enhancers did not cause perinatal death, mice that survived the deletions did show signs of developmental defects after more comprehensively inspecting for phenotypic changes under different conditions. For example, deleting a conserved and Shh regulating enhancer resulted in degenerations of skeletal elements in limb bud [48] and deleting an ultra-conserved limb-developmental associated enhancer led to significantly decreased body size in mouse embryos [49]. Dickel et al showed that single enhancer deletions of three out of the four enhancers regulating the Aristaless-related homeobox (ARX/Arx) gene led to decreased overall growth or brain abnormality in transgenic mice [50].…”

Conserved non-coding elements: developmental gene regulation meets genome organization

“…Mutations in a CNE proximal to the HMX1 gene cause aberrant external ear development in wild populations of rats and highland cattle ( 39 ). A mouse sequence called M280 , which contains a CNE identical between human, mouse and rat, is indispensable for body growth in mice ( 40 ). Many more cases linking CNEs to both human disease and lineage-specific traits are discussed in more depth in the ‘ Diseases associated with non-coding conservation’ and ‘ CNE Modifications and Losses ’ sections.…”