Diphthamide is a unique post-translationally modified histidine residue (His 715 in all mammals) found only in eukaryotic elongation factor-2 (eEF-2). The biosynthesis of diphthamide represents one of the most complex modifications, executed by protein factors conserved from yeast to humans. Diphthamide is not only essential for normal physiology (such as ensuring fidelity of mRNA translation), but is also exploited by bacterial ADP-ribosylating toxins (e.g., diphtheria toxin) as their molecular target in pathogenesis. Taking advantage of the observation that cells defective in diphthamide biosynthesis are resistant to ADP-ribosylating toxins, in the past four decades, seven essential genes ( Dph1 to Dph7) have been identified for diphthamide biosynthesis. These technically unsaturated screens raise the question as to whether additional genes are required for diphthamide biosynthesis. In this study, we performed two independent, saturating, genome-wide CRISPR knockout screens in human cells. These screens identified all previously known Dph genes, as well as further identifying the BTB/POZ domain-containing transcription factor Miz1. We found that Miz1 is absolutely required for diphthamide biosynthesis via its role in the transcriptional regulation of Dph1 expression. Mechanistically, Miz1 binds to the Dph1 proximal promoter via an evolutionarily conserved consensus binding site to activate Dph1 transcription. Therefore, this work demonstrates that Dph1-7, along with the newly identified Miz1 transcription factor, are likely to represent the essential protein factors required for diphthamide modification on eEF2. Author summary Diphthamide is a unique post-translationally modified histidine residue (His 699 in yeast, His 715 in all mammals) found only in eukaryotic elongation factor-2 (eEF-2). Mice that are deficient in diphthamide biosynthesis are embryonic lethal, attesting to the importance of diphthamide in normal physiology. It has taken four decades to identify the seven non-redundant genes in diphthamide biosynthesis, but whether additional factors are required and how the pathway is regulated remained elusive. To address these issues, we performed two saturating, independent, and unbiased genome-wide CRISPR knockout screens. The screens concluded independently that Dph1-Dph7 and additionally transcription factor Miz1 are the key factors required for diphthamide biosynthesis. Mechanistically, Miz1 binds to the Dph1 proximal promoter via an evolutionarily conserved consensus binding site to activate Dph1 transcription. While diphthamide biosynthesis machinery (Dph1-Dph7) exists across eukaryotes, Miz1 orthologues do not exist in lower species such as yeast, C. elegans, and Drosophila, indicating that the regulation of diphthamide modification by Miz1 emerged much later in evolution. The work opens a new avenue for understanding the role that diphthamide modification plays in normal physiology and bacterial toxin pathogenesis.
【초록키워드】 Evolution, Pathogenesis, Transcription, binding site, Protein, CRISPR, mice, humans, pathway, expression, yeast, Bacterial, mRNA translation, Biosynthesis, transcription factor, histidine, opens, bacterial toxin, observation, Drosophila, Consensus, Factor, Regulation, complex, residue, transcriptional regulation, human cells, molecular target, key factor, modifications, mammals, Modification, eukaryotes, elegans, promoter, Diphthamide, DPH1, Dph7, eEF2, toxin, toxins, embryonic lethal, while, Genes, Cell, independent, Seven, bind, raise, identify, performed, conserved, remained, required, question, unique, regulated, activate, eukaryotic, proximal, Taking, orthologue, unsaturated, 【제목키워드】 identification, transcription factor,