RESUMO
X-linked acrogigantism (X-LAG) is the most severe form of pituitary gigantism and is characterized by aggressive growth hormone (GH)-secreting pituitary tumors that occur in early childhood. X-LAG is associated with chromosome Xq26.3 duplications (the X-LAG locus typically includes VGLL1, CD40LG, ARHGEF6, RBMX, and GPR101) that lead to massive pituitary tumoral expression of GPR101, a novel regulator of GH secretion. The mechanism by which the duplications lead to marked pituitary misexpression of GPR101 alone was previously unclear. Using Hi-C and 4C-seq, we characterized the normal chromatin structure at the X-LAG locus. We showed that GPR101 is located within a topologically associating domain (TAD) delineated by a tissue-invariant border that separates it from centromeric genes and regulatory sequences. Next, using 4C-seq with GPR101, RBMX, and VGLL1 viewpoints, we showed that the duplications in multiple X-LAG-affected individuals led to ectopic interactions that crossed the invariant TAD border, indicating the existence of a similar and consistent mechanism of neo-TAD formation in X-LAG. We then identified several pituitary active cis-regulatory elements (CREs) within the neo-TAD and demonstrated in vitro that one of them significantly enhanced reporter gene expression. At the same time, we showed that the GPR101 promoter permits the incorporation of new regulatory information. Our results indicate that X-LAG is a TADopathy of the endocrine system in which Xq26.3 duplications disrupt the local chromatin architecture forming a neo-TAD. Rewiring GPR101-enhancer interaction within the new regulatory unit is likely to cause the high levels of aberrant expression of GPR101 in pituitary tumors caused by X-LAG.
Assuntos
Acromegalia , Doenças Genéticas Ligadas ao Cromossomo X , Gigantismo , Neoplasias Hipofisárias , Acromegalia/complicações , Acromegalia/genética , Acromegalia/patologia , Pré-Escolar , Cromatina/genética , Comunicação , Proteínas de Ligação a DNA/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Gigantismo/complicações , Gigantismo/genética , Gigantismo/patologia , Humanos , Neoplasias Hipofisárias/genética , Receptores Acoplados a Proteínas G/genética , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: The organisation of vertebrate genomes into topologically associating domains (TADs) is believed to facilitate the regulation of the genes located within them. A remaining question is whether TAD organisation is achieved through the interactions of the regulatory elements within them or if these interactions are favoured by the pre-existence of TADs. If the latter is true, the fusion of two independent TADs should result in the rewiring of the transcriptional landscape and the generation of ectopic contacts. RESULTS: We show that interactions within the PAX3 and FOXO1 domains are restricted to their respective TADs in normal conditions, while in a patient-derived alveolar rhabdomyosarcoma cell line, harbouring the diagnostic t(2;13)(q35;q14) translocation that brings together the PAX3 and FOXO1 genes, the PAX3 promoter interacts ectopically with FOXO1 sequences. Using a combination of 4C-seq datasets, we have modelled the three-dimensional organisation of the fused landscape in alveolar rhabdomyosarcoma. CONCLUSIONS: The chromosomal translocation that leads to alveolar rhabdomyosarcoma development generates a novel TAD that is likely to favour ectopic PAX3:FOXO1 oncogene activation in non-PAX3 territories. Rhabdomyosarcomas may therefore arise from cells which do not normally express PAX3. The borders of this novel TAD correspond to the original 5'- and 3'- borders of the PAX3 and FOXO1 TADs, respectively, suggesting that TAD organisation precedes the formation of regulatory long-range interactions. Our results demonstrate that, upon translocation, novel regulatory landscapes are formed allowing new intra-TAD interactions between the original loci involved.
Assuntos
Proteína Forkhead Box O1/genética , Fator de Transcrição PAX3/genética , Mapas de Interação de Proteínas/genética , Rabdomiossarcoma Alveolar/genética , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Proteínas de Fusão Oncogênica/genética , Regiões Promotoras Genéticas , Domínios Proteicos/genética , Sequências Reguladoras de Ácido Nucleico/genética , Rabdomiossarcoma Alveolar/patologia , Translocação Genética/genéticaRESUMO
Complex genomes utilize insulators and boundary elements to help define spatial and temporal gene expression patterns. We report that a genome-wide B1 SINE (Short Interspersed Nuclear Element) retrotransposon (B1-X35S) has potent intrinsic insulator activity in cultured cells and live animals. This insulation is mediated by binding of the transcription factors dioxin receptor (AHR) and SLUG (SNAI2) to consensus elements present in the SINE. Transcription of B1-X35S is required for insulation. While basal insulator activity is maintained by RNA polymerase (Pol) III transcription, AHR-induced insulation involves release of Pol III and engagement of Pol II transcription on the same strand. B1-X35S insulation is also associated with enrichment of heterochromatin marks H3K9me3 and H3K27me3 downstream of B1-X35S, an effect that varies with cell type. B1-X35S binds parylated CTCF and, consistent with a chromatin barrier activity, its positioning between two adjacent genes correlates with their differential expression in mouse tissues. Hence, B1 SINE retrotransposons represent genome-wide insulators activated by transcription factors that respond to developmental, oncogenic, or toxicological stimuli.