An improved functional analysis of linker-mediated complex (iFALC) strategy
| Autor: | Masayuki Seki, Masahiko Harata, Yu Nakabayashi |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
endocrine system Saccharomyces cerevisiae Proteins animal structures Recombinant Fusion Proteins Protein subunit Biophysics Saccharomyces cerevisiae medicine.disease_cause Biochemistry Cell Line Histones Gene Knockout Techniques 03 medical and health sciences 0302 clinical medicine Protein Interaction Mapping Histone H2A Histone H2B medicine Animals Molecular Biology Gene Gene knockout Mutation biology Chemistry Chromatin binding Cell Biology Chromatin Cell biology 030104 developmental biology Histone Multiprotein Complexes 030220 oncology & carcinogenesis embryonic structures biology.protein Mutant Proteins Chickens Protein Binding |
| Zdroj: | Biochemical and Biophysical Research Communications. 526:1164-1169 |
| ISSN: | 0006-291X |
| DOI: | 10.1016/j.bbrc.2020.04.039 |
| Popis: | The functional analysis of linker-mediated complex (FALC) strategy that facilitates functional analysis of a common subunit of multi-subunit protein complexes in cells constitutes three steps; (1) a common subunit is fused to a specific subunit via recombinant DNA, (2) mutation is introduced into a portion of the common subunit of the fused protein, and (3) the mutational effect on the fused protein is evaluated by transformation and analysis of multiple appropriate gene knockout yeast strains. Conceptually, the FALC strategy is applicable to any common subunit of multi-subunit protein complexes in any cell type. However, the proximity of two subunits to fuse, preparation of multiple gene knockout cells, and utilization of yeast cells can together prevent the practical and broad usage of the FALC strategy for analyzing all multi-subunit complexes in all cell types. In this study, we analyzed histone H2B as a common subunit of histone H2A/H2B and histone variant H2A.Z/H2B dimers. The FALC strategy was improved in three ways; (i) a long linker (up to 300 amino acids) was used to fuse H2B with H2A.Z in yeast cells, (ii) the effects of the fused H2B-H2A.Z harboring mutation in the H2B portion was evaluated in H2A.Z knockout yeast strains and it was not essential to knockout two copies of H2B genes, and (iii) this occurred even in vertebrate cells possessing a dozen H2B genes. This improved FALC (iFALC) strategy reveals that vertebrate H2B-D68, corresponding to yeast H2B-D71, is critical for chromatin binding of the H2A.Z/H2B dimer, and this is evolutionarily conserved. |
| Databáze: | OpenAIRE |
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