Detection and Quantification of Histone Methyltransferase Activity In Vitro.

Histone methyltransferases (HMTs) catalyze the methylation of lysine and arginine residues in histone as well as nonhistone substrates. In vitro histone methyltransferase assays have been instrumental in identifying HMTs, and they continue to be invaluable tools for the study of these important enzymes, revealing novel substrates and modes of regulation.Here we describe a universal protocol to examine HMT activity in vitro that can be adapted to a range of HMTs, substrates, and experimental objectives. We provide protocols for the detection of activity based on incorporation of 3H-labeled methyl groups from S-adenosylmethionine (SAM), methylation-specific antibodies, and quantification of the reaction product S-adenosylhomocysteine (SAH).

Translation elongation factor 1A2 is encoded by one of four closely related eef1a genes and is dispensable for survival in zebrafish.

Zebrafish are valuable model organisms for the study of human single-gene disorders: they are genetically manipulable, their development is well understood, and mutant lines with measurable, disease-appropriate phenotypic abnormalities can be used for high throughput drug screening approaches. However, gene duplication events in zebrafish can result in redundancy of gene function, masking loss-of-function phenotypes and thus confounding this approach to disease modelling. Furthermore, recent studies have yielded contrasting results depending on whether specific genes are targeted using genome editing to make mutant lines, or whether morpholinos are used (morphants). De novo missense mutations in the human gene EEF1A2, encoding a tissue-specific translation elongation factor, cause severe neurodevelopmental disorders; there is a real need for a model system to study these disorders and we wanted to explore the possibility of a zebrafish model. We identified four eef1a genes and examined their developmental and tissue-specific expression patterns: eef1a1l1 is first to be expressed while eef1a2 is only detected later during development. We then determined the effects of introducing null mutations into translation elongation factor 1A2 (eEF1A2) in zebrafish using CRISPR/Cas9 gene editing, in order to compare the results with previously described morphants, and with severe neurodegenerative lethal phenotype of eEF1A2-null mice. In contrast with both earlier analyses in zebrafish using morpholinos and with the mouse eEF1A2-null mice, disruption of the eef1a2 gene in zebrafish is compatible with normal lifespan. The resulting lines, however, may provide a valuable platform for studying the effects of expression of mutant human eEF1A2 mRNA.