Electrophoretic mobility shift assays with GFP-tagged proteins (GFP-EMSA)

The electrophoretic mobility shift assay (EMSA) is commonly used for the study of nucleic acid-binding proteins. The technique can be used to demonstrate that a protein is binding to RNA or DNA through visualization of a shift in electrophoretic mobility of the nucleic acid band. A major disadvantage of the EMSA is that it does not always provide an absolute certitude that the band shift is due to the protein under scrutiny, as contaminants in the sample could also cause the band shift. Here we describe a variation of the standard EMSA allowing to visualize with added certitude, the co-localized band shifts of a GFP-tagged protein binding to its cognate nucleic acid target sequence stained with an intercalator, such as GelRed. Herein, we present an illustrative protocol of this useful technique called GFP-EMSA along with specific notes on its advantages and limitations

Disruption of an EHMT1-associated chromatin-modification module causes intellectual disability

Item does not contain fulltextIntellectual disability (ID) disorders are genetically and phenotypically highly heterogeneous and present a major challenge in clinical genetics and medicine. Although many genes involved in ID have been identified, the etiology is unknown in most affected individuals. Moreover, the function of most genes associated with ID remains poorly characterized. Evidence is accumulating that the control of gene transcription through epigenetic modification of chromatin structure in neurons has an important role in cognitive processes and in the etiology of ID. However, our understanding of the key molecular players and mechanisms in this process is highly fragmentary. Here, we identify a chromatin-modification module that underlies a recognizable form of ID, the Kleefstra syndrome phenotypic spectrum (KSS). In a cohort of KSS individuals without mutations in EHMT1 (the only gene known to be disrupted in KSS until now), we identified de novo mutations in four genes, MBD5, MLL3, SMARCB1, and NR1I3, all of which encode epigenetic regulators. Using Drosophila, we demonstrate that MBD5, MLL3, and NR1I3 cooperate with EHMT1, whereas SMARCB1 is known to directly interact with MLL3. We propose a highly conserved epigenetic network that underlies cognition in health and disease. This network should allow the design of strategies to treat the growing group of ID pathologies that are caused by epigenetic defects