| Post-translational modification(PTM)of histones is widely recognized as one of the fundamental mechanisms of epigenetic regulation of biological processes.To date,more than 500 chemically distinct histone PTMs have been identified,including those that play a role in diseases from cancer to neurological disorders.However,regardless of this significance,our knowledge of the identity of the proteins that are indirectly or directly responsible for the modification,i.e.,the regulators,the “writers” and “erasers” that regulate,add and remove,respectively,the modification – is quite poor.Several existing strategies for the discovery of the regulators are either based on structure and sequence similarity or relied on gene mutant detection.An efficient strategy is wanted for fast and global identification of regulators at an affordable cost.Thus,based on our knowledge of protein microarray technology,we anticipate to develop a novel technique,which takes yeast as a model,combining protein microarray and histone modification antibodies,for global,de novo identification of regulators of histone PTMs.Firstly,we carried out western blot and dot blot to screen for specific antibodies.Six antibodies specific to histone marks passed our selection criterion.Then we optimized printing performance by testing three types of 3-dimensional polymer coating slides and different lysate buffers.After that,we prepared a massive cell-lysate microarray from 4,837 yeast knockout(YKO)strains and 322 temperature-sensitive mutant strains(for essential genes),and probed this microarray for changes in the degree of a specific histone modification.Mutant strains with reduced modification identified proteins important for “writing”;those with enhanced modification identified proteins for “erasing”.Reflecting the fact that this assay probes thousands of different strains in a single assay,we named our method,cell lysate microarray on kilo-conditions(CLICK).To validate this technique,we first investigated the regulators of two wellknown modifications,i.e.,H3K4me3 and H3K36me3.The results showed that,in just a single experiment,this method identified 80% of the previously known regulators of these modifications that had been discovered in the last 10 years.In addition,comparing the results of H3K4me3 and H3K36me3,we firstly suggested a strong crosstalk between H3K4me3 and H3K36me3,which was mediated by Set2 p,through their different roles in histone acetylation regulation.Given the capability of CLICK array to identify the regulators of wellstudied modifications,we next applied this method to the far less studied H4K16 ac modification,and unexpectedly identified Cab4 p and Cab5 p,the last two enzymes in the CoA biosynthesis pathway,as fundamentally critical proteins involved in “writing” H4K16 acetylation.We showed that these two enzymes also play an essential role in not just histone acetylation,but also histone butyrylation,propionylation,crotonylation,and ?-hydroxybutyrylation: in short,all histone acylations that we had tested.As the CoA biosynthesis pathway was conserved from bacteria to mammalian,the homologous protein in human,COASY,may cause a similar depletion of histone acylation.In summary,in this study,taking advantage of the high-density array format and the specific antibodies,we established the CLICK array strategy.The CLICK array offers a simple,unbiased,and physiological relevant method to search for novel regulators on a proteome scale without prior assumptions.We believe that this approach could be widely adopted to discover new members of regulators for a variety of histone marks.Additionally,it could also be the method-of-choice to rapidly identify an upstream protein/enzyme(s)that regulate/modify(either add or remove)the level of a protein or a novel protein posttranslational modification as discovered by recent efforts in mass spectrometry. |
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