Identification of the transcription factor Miz1 as an essential regulator of diphthamide biosynthesis using a CRISPR-mediated genome-wide screen

Autoři: Jie Liu aff001;  Zehua Zuo aff001;  Meijuan Zou aff001;  Toren Finkel aff001;  Shihui Liu aff001
Působiště autorů: Aging Institute of University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh, PA, United States of America aff001;  Division of Cardiology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States of America aff002;  Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States of America aff003
Vyšlo v časopise: Identification of the transcription factor Miz1 as an essential regulator of diphthamide biosynthesis using a CRISPR-mediated genome-wide screen. PLoS Genet 16(10): e32767. doi:10.1371/journal.pgen.1009068
Kategorie: Research Article
doi: 10.1371/journal.pgen.1009068


Diphthamide is a unique post-translationally modified histidine residue (His715 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.

Klíčová slova:

Biosynthesis – CRISPR – DNA methylation – Genetic screens – Library screening – Toxins – Transfection – HT1080 cells


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