A Novel Auto-catalysis/Two-Hybrid System To Identify Protein-Protein Interactions Involving Post-Translational Modifications

Core histones H2A,H2B,H3 and H4 are the essential proteins for chromatin organization and cell viability. DNA-templated nuclear activities, including transcription, replication, recombination, and repair, all take place within the chromatin. Two molecules of each of the core histones are wrapped around by about 150 base pairs of DNA to form a nucleosome. Formation of nucleosomes requires extensive histone-histone and histone-DNA interactions occurred mainly within the central histone-fold domain of each core histone. The N' termini of all core histones are "flexible tails" which protrude from the nucleosomal core particles and are largely unstructured. The yeast cells without histones H3 and H4, or H2A and H2B N' termini are not viable ,they die at G2/M phase of the cell cycle. Histone tails are subject to multiple post-translational modifications which include acetylation, methylation, phosphorylation, ubiquitylation , sumolation etc.Many of these modifications may change the ionic charge of the highly basic histones, which have substantial effects on the compact structure of chromatin. Usually the compact structure of chromatin restricts the binding and progression of protein factors. The acetylated histone tail peptide interacts with general DNA at much more higher Km than that of unacetylated ,that means a weakened DNA histone binding interaction may allow for better access of DNA binding and processing factors to find their cognate DNA elements. Generally speaking, histone hyper- and hypo-acetylation each triggers transcriptional activation and repression / silence, respectively. Acetylated histones have been shown to be bound by a highly conserved motif, bromodomain,that is found in many transcriptional activators. Likewise, methylated histones recruit chromodomain, that is frequently found to be part of transcriptional repressors. In addition to acetylation, the other modification pattern including phosporylation, methylation and ubiquitylation were found to be involved in gene specific regulation. In addition, different histone modifications may influence each other. for instance, acetylation at lysine 14 of H3 by several histone acetyltransferases (HAT) has been shown to be facilitated by phosphorylation at serine 10, and there is same case between arginine 3 methlylation of H4 and lysine 8 acetylation.These evidences has caused scientists to favor the view that each histone modification pattern may be read by other proteins or protein modules, and thus followed different biological procedures. In 2000, David Allis proposed the "histone code" hypothesis which suggests that differentially modified histones may act as specific signals that are interpreted by the binding of selective factors. Such factors thus perform certain functions at the genomic loci bearing the corresponding histone modifications. Thus far, all modified histone-binding proteins were identified by biochemical methods. Genome-wide screening for proteins possessing affinity toward specifically modified histones needs to be done. To identify the modified histone binding proteins, previous strategies have been based on the ectopic expression of protein kinases in the two-hybrid hosts which normally lack such enzymes. For example, tyrosine kinases and mammalian serine/threonine kinases were respectively expressed in yeast and E. coli for two-hybrid screens. The substrate proteins to be screened in these assays were phosphorylated and hence allowed the detection of interactions involving these phosphorylation events. Major concerns of these methods include the efficiency and specificity with which the bait protein to be screened is modified. Phosphorylation relies on a typical trans reaction between an enzyme and its substrates. If the foreign enzyme cannot identify the hybrid protein in the sea of host cellular proteins for efficient modification, the subsequent screen is likely to be unsuccessful. The efficiency of modification may be increased by overexpressing the enzyme, but inadvertent modifications of host protein...