Search for: All records

Chemical tools to control the activities and interactions of chromatin components have broad impact on our understanding of cellular and disease processes. It is important to accurately identify their molecular effects to inform clinical efforts and interpretations of scientific studies. Chaetocin is a widely used chemical that decreases H3K9 methylation in cells. It is frequently attributed as a specific inhibitor of the histone methyltransferase activities of SUV39H1/SU(VAR)3–9, although prior observations showed chaetocin likely inhibits methyltransferase activity through covalent mechanisms involving its epipolythiodixopiperazine disulfide ‘warhead’ functionality. The continued use of chaetocin in scientific studies may derive from the net effect of reduced H3K9 methylation, irrespective of a direct or indirect mechanism. However, there may be other molecular impacts of chaetocin on SUV39H1 besides inhibition of H3K9 methylation levels that could confound the interpretation of past and future experimental studies. Here, we test a new hypothesis that chaetocin may have an additional downstream impact aside from inhibition of methyltransferase activity. Using a combination of truncation mutants, a yeast two-hybrid system, and direct in vitro binding assays, we show that the human SUV39H1 chromodomain (CD) and HP1 chromoshadow domain (CSD) directly interact. Chaetocin inhibits this binding interaction through its disulfide functionality with some specificity by covalently binding with the CD of SUV39H1, whereas the histone H3–HP1 interaction is not inhibited. Given the key role of HP1 dimers in driving a feedback cascade to recruit SUV39H1 and to establish and stabilize constitutive heterochromatin, this additional molecular consequence of chaetocin should be broadly considered. 

Histone post-translational modifications are small chemical changes to the histone protein structure that have cascading effects on diverse cellular functions. Detecting histone modifications and characterizing their binding partners are critical steps in understanding chromatin biochemistry and have been accessed using common reagents such as antibodies, recombinant assays, and FRET-based systems. High-throughput platforms could accelerate work in this field, and also could be used to engineer de novo histone affinity reagents; yet, published studies on their use with histones have been noticeably sparse. Here, we describe specific experimental conditions that affect binding specificities of post-translationally modified histones in classic protein engineering platforms and likely explain the relative difficulty with histone targets in these platforms. We also show that manipulating avidity of binding interactions may improve specificity of binding. 

Abstract UBE3A is an E3 ubiquitin ligase encoded by the neurally imprinted UBE3A gene. The abundance and subcellular distribution of UBE3A has been the topic of many previous studies as its dosage and localization has been linked to neurodevelopmental disorders including Autism, Dup15q syndrome, and Angelman syndrome. While commercially available antibodies have been widely employed to determine UBE3A localization, an extensive analysis and comparison of the performance of different UBE3A antibodies has not been conducted. Here we evaluated the specificities of seven commercial UBE3A antibodies in two of the major experimental models used in UBE3A research, mouse and human pluripotent stem cell-derived neural cells and tissues. We tested these antibodies in their two most common assays, immunofluorescence and western blot. In addition, we also assessed the ability of these antibodies to capture dynamic spatiotemporal changes of UBE3A by utilizing human cerebral organoid models. Our results reveal that among the seven antibodies tested, three antibodies demonstrated substantial nonspecific immunoreactivity. While four antibodies show specific localization patterns in both mouse brain sections and human cerebral organoids, these antibodies varied significantly in background signals and staining patterns in undifferentiated human pluripotent stem cells.