• intrabody
• PTM
• histone
• HAT

Post-translational modifications (PTM) of proteins are crucial in many cellular processes including chromatin remodeling, transcriptional regulation and cellular signaling.
A PTM-modified protein can play a very different role, compared to the non-modified version of the same protein. For this reason, PTMs represent a relevant biological target, and functional studies of PTM-proteins will allow to dissect in vivo complex cellular pathways.
However, no general method currently is available to selectively target a post-translationally modified version of a given protein target. Indeed, the more common approaches to study protein function in vivo, such as RNA interference, gene KO or genome editing with CRISP-Cas9, operate only at an upstream level and as a consequence result inefficient at selectively interfering with the modified version of a protein.
One indirect method for targeting PTMs, that is currently being pursued, consists in the use of small molecules that are able to inhibit the activity of the enzymes responsible for the PTM itself. However, this class of inhibitors generate broad-spectrum effects, due to the poor specificity of some inhibitors and, more crucially, to the multiplicity of the enzyme substrate targets that are naturally modified, so it becomes almost impossible to have a specific effect only on the modified protein of interest.
Thus the development of experimental strategies and tools for the selective interference of PTM-proteins could be crucial for research and clinical perspectives.
One such tool is represented by intrabodies, engineered antibody domains that have been proved to specifically perturb the function of intracellular antigens.
A recent technology, named P.I.S.A (Post-translational Intracellular Silencing Antibody), allows to select intracellular antibodies that show binding specificity to a defined PTM-protein, representing a turning point on the pathway of protein interference.
This technology is based on a platform for two-hybrid selection in yeast, known as IACT (Intracellular Antibody Capture Technology) combined with Tethered Catalysis, which enable the specific selection of antibody-antigen interactions based on the presence of PTMs.
In previous work in the lab, this technique yielded an intrabody that specifically and selectively binds the acetylated form of histone H3.
The aim of this thesis project is to characterize the silencing action of this specific intrabody evaluating the downstream effects on global gene expression and to compare this action to that obtained with the use of drugs that inhibit histone acetyl-transferases (HATs), using the Saccharomyces cerevisiae yeast as a model organism.
This objective will be pursued by comparing the different experimental conditions through RNA-seq analysis, evaluating the different global gene expression patterns.