RESUMO
Genomic context critically modulates regulatory function but is difficult to manipulate systematically. The murine insulin-like growth factor 2 (Igf2)/H19 locus is a paradigmatic model of enhancer selectivity, whereby CTCF occupancy at an imprinting control region directs downstream enhancers to activate either H19 or Igf2. We used synthetic regulatory genomics to repeatedly replace the native locus with 157-kb payloads, and we systematically dissected its architecture. Enhancer deletion and ectopic delivery revealed previously uncharacterized long-range regulatory dependencies at the native locus. Exchanging the H19 enhancer cluster with the Sox2 locus control region (LCR) showed that the H19 enhancers relied on their native surroundings while the Sox2 LCR functioned autonomously. Analysis of regulatory DNA actuation across cell types revealed that these enhancer clusters typify broader classes of context sensitivity genome wide. These results show that unexpected dependencies influence even well-studied loci, and our approach permits large-scale manipulation of complete loci to investigate the relationship between regulatory architecture and function.
Assuntos
Fator de Ligação a CCCTC , Elementos Facilitadores Genéticos , Fator de Crescimento Insulin-Like II , RNA Longo não Codificante , Fatores de Transcrição SOXB1 , Animais , Camundongos , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Região de Controle de Locus Gênico/genética , Impressão Genômica , Genômica/métodosRESUMO
Pervasive transcriptional activity is observed across diverse species. The genomes of extant organisms have undergone billions of years of evolution, making it unclear whether these genomic activities represent effects of selection or 'noise'1-4. Characterizing default genome states could help understand whether pervasive transcriptional activity has biological meaning. Here we addressed this question by introducing a synthetic 101-kb locus into the genomes of Saccharomyces cerevisiae and Mus musculus and characterizing genomic activity. The locus was designed by reversing but not complementing human HPRT1, including its flanking regions, thus retaining basic features of the natural sequence but ablating evolved coding or regulatory information. We observed widespread activity of both reversed and native HPRT1 loci in yeast, despite the lack of evolved yeast promoters. By contrast, the reversed locus displayed no activity at all in mouse embryonic stem cells, and instead exhibited repressive chromatin signatures. The repressive signature was alleviated in a locus variant lacking CpG dinucleotides; nevertheless, this variant was also transcriptionally inactive. These results show that synthetic genomic sequences that lack coding information are active in yeast, but inactive in mouse embryonic stem cells, consistent with a major difference in 'default genomic states' between these two divergent eukaryotic cell types, with implications for understanding pervasive transcription, horizontal transfer of genetic information and the birth of new genes.
Assuntos
Genes Sintéticos , Genoma , Saccharomyces cerevisiae , Transcrição Gênica , Animais , Humanos , Camundongos , Cromatina/genética , Ilhas de CpG , Genes Sintéticos/genética , Genoma/genética , Células-Tronco Embrionárias Murinas/metabolismo , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/genética , Hipoxantina Fosforribosiltransferase/genética , Evolução MolecularRESUMO
The loss of the tail is among the most notable anatomical changes to have occurred along the evolutionary lineage leading to humans and to the 'anthropomorphous apes'1-3, with a proposed role in contributing to human bipedalism4-6. Yet, the genetic mechanism that facilitated tail-loss evolution in hominoids remains unknown. Here we present evidence that an individual insertion of an Alu element in the genome of the hominoid ancestor may have contributed to tail-loss evolution. We demonstrate that this Alu element-inserted into an intron of the TBXT gene7-9-pairs with a neighbouring ancestral Alu element encoded in the reverse genomic orientation and leads to a hominoid-specific alternative splicing event. To study the effect of this splicing event, we generated multiple mouse models that express both full-length and exon-skipped isoforms of Tbxt, mimicking the expression pattern of its hominoid orthologue TBXT. Mice expressing both Tbxt isoforms exhibit a complete absence of the tail or a shortened tail depending on the relative abundance of Tbxt isoforms expressed at the embryonic tail bud. These results support the notion that the exon-skipped transcript is sufficient to induce a tail-loss phenotype. Moreover, mice expressing the exon-skipped Tbxt isoform develop neural tube defects, a condition that affects approximately 1 in 1,000 neonates in humans10. Thus, tail-loss evolution may have been associated with an adaptive cost of the potential for neural tube defects, which continue to affect human health today.