High-Throughput Protein Production Combined with High- Throughput SELEX Identifies an Extensive Atlas of Ciona robusta Transcription Factor DNA-Binding Specificities

International audienceTranscription factors (TFs) control gene transcription, binding to specific DNA motifs located in cis-regulatory elements across the genome. The identification of TF-binding motifs is thus an important aspect to understand the role of TFs in gene regulation. SELEX, Systematic Evolution of Ligands by EXponential enrichment, is an efficient in vitro method, which can be used to determine the DNA-binding specificity of TFs. Thanks to the development of high-throughput (HT) DNA cloning system and protein production technology, the classical SELEX assay has be extended to high-throughput scale (HT-SELEX).We report here the detailed protocol for the cloning, production, and purification of 420 Ciona robusta DNA BD. 263 Ciona robusta TF DNA-binding domain proteins were purified in milligram quantities and analyzed by HT-SELEX. The identification of 139 recognition sequences generates an atlas of protein-DNA-binding specificities that is crucial for the understanding of the gene regulatory network (GRN) of Ciona robusta. Overall, our analysis suggests that the Ciona robusta repertoire of sequence-specific transcription factors comprises less than 500 genes. The protocols for high-throughput protein production and HT-SELEX described in this article for the study of Ciona robusta TF DNA-binding specificity are generic and have been successfully applied to a wide range of TFs from other species, including human, mouse, and Drosophila

De novo SMARCA2 variants clustered outside the helicase domain cause a new recognizable syndrome with intellectual disability and blepharophimosis distinct from Nicolaides–Baraitser syndrome

Purpose: Nontruncating variants in SMARCA2, encoding a catalytic subunit of SWI/SNF chromatin remodeling complex, cause Nicolaides\u2013Baraitser syndrome (NCBRS), a condition with intellectual disability and multiple congenital anomalies. Other disorders due to SMARCA2 are unknown. Methods: By next-generation sequencing, we identified candidate variants in SMARCA2 in 20 individuals from 18 families with a syndromic neurodevelopmental disorder not consistent with NCBRS. To stratify variant interpretation, we functionally analyzed SMARCA2 variants in yeasts and performed transcriptomic and genome methylation analyses on blood leukocytes. Results: Of 20 individuals, 14 showed a recognizable phenotype with recurrent features including epicanthal folds, blepharophimosis, and downturned nasal tip along with variable degree of intellectual disability (or blepharophimosis intellectual disability syndrome [BIS]). In contrast to most NCBRS variants, all SMARCA2 variants associated with BIS are localized outside the helicase domains. Yeast phenotype assays differentiated NCBRS from non-NCBRS SMARCA2 variants. Transcriptomic and DNA methylation signatures differentiated NCBRS from BIS and those with nonspecific phenotype. In the remaining six individuals with nonspecific dysmorphic features, clinical and molecular data did not permit variant reclassification. Conclusion: We identified a novel recognizable syndrome named BIS associated with clustered de novo SMARCA2 variants outside the helicase domains, phenotypically and molecularly distinct from NCBRS