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

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.

Structural basis for dimerization quality control.

Most quality control pathways target misfolded proteins to prevent toxic aggregation and neurodegeneration1. Dimerization quality control further improves proteostasis by eliminating complexes of aberrant composition2, but how it detects incorrect subunits remains unknown. Here we provide structural insight into target selection by SCF-FBXL17, a dimerization-quality-control E3 ligase that ubiquitylates and helps to degrade inactive heterodimers of BTB proteins while sparing functional homodimers. We find that SCF-FBXL17 disrupts aberrant BTB dimers that fail to stabilize an intermolecular ß-sheet around a highly divergent ß-strand of the BTB domain. Complex dissociation allows SCF-FBXL17 to wrap around a single BTB domain, resulting in robust ubiquitylation. SCF-FBXL17 therefore probes both shape and complementarity of BTB domains, a mechanism that is well suited to establish quality control of complex composition for recurrent interaction modules.

BTB/POZ zinc finger protein ZBTB16 inhibits breast cancer proliferation and metastasis through upregulating ZBTB28 and antagonizing BCL6/ZBTB27.

BACKGROUND: Breast cancer remains in urgent need of reliable diagnostic and prognostic markers. Zinc finger and BTB/POZ domain-containing family proteins (ZBTBs) are important transcription factors functioning as oncogenes or tumor suppressors. The role and regulation of ZBTB16 in breast cancer remain to be established. METHODS: Reverse-transcription PCR and methylation-specific PCR were applied to detect expression and methylation of ZBTB16 in breast cancer cell lines and tissues. The effects of ZBTB16 in breast cancer cells were examined via cell viability, CCK8, Transwell, colony formation, and flow cytometric assays. Xenografts and immunohistochemistry analyses were conducted to determine the effects of ZBTB16 on tumorigenesis in vivo. The specific mechanisms of ZBTB16 were further investigated using Western blot, qRT-PCR, luciferase assay, and co-IP. RESULTS: ZBTB16 was frequently downregulated in breast cancer cell lines in correlation with its promoter CpG methylation status. Restoration of ZBTB16 expression led to induction of G2/M phase arrest and apoptosis, inhibition of migration and invasion, reversal of EMT, and suppression of cell proliferation, both in vitro and in vivo. Furthermore, ectopically expressed ZBTB16 formed heterodimers with ZBTB28 or BCL6/ZBTB27 and exerted tumor suppressor effects through upregulation of ZBTB28 and antagonistic activity on BCL6. CONCLUSIONS: Low expression of ZBTB16 is associated with its promoter hypermethylation and restoration of ZBTB16 inhibits tumorigenesis. ZBTB16 functions as a tumor suppressor through upregulating ZBTB28 and antagonizing BCL6. Our findings also support the possibility of ZBTB16 being a prognostic biomarker for breast cancer.

Identification of Novel Alzheimer's Disease Loci Using Sex-Specific Family-Based Association Analysis of Whole-Genome Sequence Data.

With the advent of whole genome-sequencing (WGS) studies, family-based designs enable sex-specific analysis approaches that can be applied to only affected individuals; tests using family-based designs are attractive because they are completely robust against the effects of population substructure. These advantages make family-based association tests (FBATs) that use siblings as well as parents especially suited for the analysis of late-onset diseases such as Alzheimer's Disease (AD). However, the application of FBATs to assess sex-specific effects can require additional filtering steps, as sensitivity to sequencing errors is amplified in this type of analysis. Here, we illustrate the implementation of robust analysis approaches and additional filtering steps that can minimize the chances of false positive-findings due to sex-specific sequencing errors. We apply this approach to two family-based AD datasets and identify four novel loci (GRID1, RIOK3, MCPH1, ZBTB7C) showing sex-specific association with AD risk. Following stringent quality control filtering, the strongest candidate is ZBTB7C (Pinter = 1.83 × 10-7), in which the minor allele of rs1944572 confers increased risk for AD in females and protection in males. ZBTB7C encodes the Zinc Finger and BTB Domain Containing 7C, a transcriptional repressor of membrane metalloproteases (MMP). Members of this MMP family were implicated in AD neuropathology.

A novel PTC mutation in the BTB domain of KLHL7 gene in two patients with Bohring-Opitz syndrome-like features.

The bric-a-brac, tramtrack and broad complex (BTB) superfamily of conserved proteins are involved in ubiquitin-proteasome system that contains the Kelch-like (KLHL) gene family. Kelch-like family member 7 (KLHL7), one of the KLHL gene family, consists of one BTB/POZ domain, one BACK domain and five or six Kelch motifs. Numerous variants in KLHL7 gene domains have been reported with Crisponi syndrome/cold-induced sweating syndrome type 1 (CS/CISS1)-like features and retinitis pigmentosa 42, and have recently been identified as causing Bohring-Opitz syndrome (BOS)-like features. We report two siblings with BOS-like phenotype with healthy parents and living in Qazvin province (Central Iran). We performed whole-exome sequencing (WES) on the older patient and Sanger sequencing was carried out for validation of potential causative variants in the close family. A novel homozygous frameshift mutation, p.(Phe83Leufs*3), was identified in the BTB domain of KLHL7 that caused a premature translation-termination codon (PTC) in the two siblings with severe developmental delay, microcephaly, facial dysmorphism, peripheral retinal and optic disc atrophy and cardiac septal defects. Our findings are in agreement with the clinical spectrum of KLHL7 mutations, which are associated with BOS-like features that reports for first time in our population.

Genome-wide identification and expression analysis of the BTB domain-containing protein gene family in tomato.

BTB (broad-complex, tramtrack, and bric-a-brac) family proteins are characterized by the presence of a protein-protein interaction BTB domain. BTB proteins have diverse functions, including transcriptional regulation, protein degradation, chromatin remodeling, and cytoskeletal regulation. However, little is known about this gene family in tomato (Solanum lycopersicum), the most important model plant for crop species. In this study, 38 BTB genes were identified based on tomato whole-genome sequence. Phylogenetic analysis of BTB proteins in tomato revealed that SlBTB proteins could be divided into at least 4 subfamilies. The SlBTB proteins contains 1-3 BTB domains, and several other types of functional domains, including KCTD (Potassium channel tetramerization domain-containing), the MATH (meprin and TRAF homology), ANK (Ankyrin repeats), NPR1 (nonexpressor of pathogenesis-related proteins1), NPH3 (Nonphototropic Hypocotyl 3), TAZ zinc finger, C-terminal Kelch, Skp1 and Arm (Armadillo/beta-catenin-like repeat) domains are also found in some tomato BTB proteins. Moreover, their expression patterns in tissues/stages, in response to different abiotic stress treatments and hormones were also investigated. This study provides the first comprehensive analysis of BTB gene family in the tomato genome. The data will undoubtedly be useful for better understanding the potential functions of BTB genes, and their possible roles in mediating hormone cross-talk and abiotic stress in tomato as well as in some other relative species.

Interactions between BTB domain of CP190 and two adjacent regions in Su(Hw) are required for the insulator complex formation.

The best-studied Drosophila insulator complex consists of two BTB-containing proteins, the Mod(mdg4)-67.2 isoform and CP190, which are recruited cooperatively to chromatin through interactions with the DNA-binding architectural protein Su(Hw). While Mod(mdg4)-67.2 interacts only with Su(Hw), CP190 interacts with many other architectural proteins. In spite of the fact that CP190 is critical for the activity of Su(Hw) insulators, interaction between these proteins has not been studied yet. Therefore, we have performed a detailed analysis of domains involved in the interaction between the Su(Hw) and CP190. The results show that the BTB domain of CP190 interacts with two adjacent regions at the N-terminus of Su(Hw). Deletion of either region in Su(Hw) only weakly affected recruiting of CP190 to the Su(Hw) sites in the presence of Mod(mdg4)-67.2. Deletion of both regions in Su(Hw) prevents its interaction with CP190. Using mutations in vivo, we found that interactions with Su(Hw) and Mod(mdg4)-67.2 are essential for recruiting of CP190 to the Su(Hw) genomic sites.

Broad-complex, tramtrack, and bric-à-brac (BTB) proteins: Critical regulators of development.

Broad-complex, Tramtrack, and Bric-à-brac/poxvirus and zinc finger (BTB/POZ) family proteins are a diverse family of proteins that are characterized by the presence of a common protein-protein interaction domain, known as the BTB domain. BTB proteins have been identified in poxviruses and many eukaryotes, and have diverse functions, ranging from transcriptional regulation and chromatin remodeling to protein degradation and cytoskeletal regulation. Specificity of function is determined in part by additional domains present in BTB family proteins, as well as by interaction partners. Studies of BTB proteins in Drosophila and mammalian systems have revealed the importance of these genes in multiple developmental contexts, as well as in cancer and neurological and musculoskeletal diseases. In this review, we discuss the functions of BTB/POZ proteins during development with an emphasis on BTB-zinc finger (BTB-ZF) proteins, which play critical roles in transcriptional regulation and chromatin remodeling.

The Occurrence of the Holometabolous Pupal Stage Requires the Interaction between E93, Krüppel-Homolog 1 and Broad-Complex.

Complete metamorphosis (Holometaboly) is a key innovation that underlies the spectacular success of holometabolous insects. Phylogenetic analyses indicate that Holometabola form a monophyletic group that evolved from ancestors exhibiting hemimetabolous development (Hemimetaboly). However, the nature of the changes underlying this crucial transition, including the occurrence of the holometabolan-specific pupal stage, is poorly understood. Using the holometabolous beetle Tribolium castaneum as a model insect, here we show that the transient up-regulation of the anti-metamorphic Krüppel-homolog 1 (TcKr-h1) gene at the end of the last larval instar is critical in the formation of the pupa. We find that depletion of this specific TcKr-h1 peak leads to the precocious up-regulation of the adult-specifier factor TcE93 and, hence, to a direct transformation of the larva into the adult form, bypassing the pupal stage. Moreover, we also find that the TcKr-h1-dependent repression of TcE93 is critical to allow the strong up-regulation of Broad-complex (TcBr-C), a key transcription factor that regulates the correct formation of the pupa in holometabolous insects. Notably, we show that the genetic interaction between Kr-h1 and E93 is also present in the penultimate nymphal instar of the hemimetabolous insect Blattella germanica, suggesting that the evolution of the pupa has been facilitated by the co-option of regulatory mechanisms present in hemimetabolan metamorphosis. Our findings, therefore, contribute to the molecular understanding of insect metamorphosis, and indicate the evolutionary conservation of the genetic circuitry that controls hemimetabolan and holometabolan metamorphosis, thereby shedding light on the evolution of complete metamorphosis.

Non-canonical dynamic mechanisms of interaction between the p66Shc protein and Met receptor.

Met receptor tyrosine kinase (RTK) is known to bind to the three distinct protein isoforms encoded by the ShcA (Shc) gene. Structure-function studies have unveiled critical roles for p52Shc-dependent signalling pathways in Met-regulated biological functions. The molecular basis of the interaction between the Met and p52Shc proteins is well-defined, but not for the longest protein isoform, p66Shc. In the present study, co-immunoprecipitation assays were performed in human embryonic kidney 293 (HEK293) cells, transiently co-transfected with Met and p66Shc mutants, in order to define the molecular determinants involved in mediating Met-p66Shc interaction. Our results show that p66Shc interacts constitutively with the receptor Met, and the Grb2 (growth factor receptor-bound protein-2) and Gab1 (Grb2-associated binder-1) adaptor proteins. Although its phosphotyrosine-binding domain (PTB) and Src homology 2 (SH2) domains co-ordinate p66Shc binding to non-activated Met receptor, these phosphotyrosine-binding modules, and its collagen homology domain 2 (CH2) region, exert negative constraints. In contrast, p66Shc interaction with the activated Met depends mainly on the integrity of its PTB domain, and to a lesser extent of its SH2 domain. Even though not required for the recruitment of p66Shc, tyrosine phosphorylation of p66Shc by activated Met enhances these interactions by mechanisms not reliant on the integrity of the Met multisubstrate-binding site. In turn, this increases phosphotyrosine-dependent p66Shc-Grb2-Gab1 complex formation away from the receptor, while blocking Grb2 and Gab1 recruitment to activated Met. In conclusion, we identify, for the first time, a novel non-canonical dynamic mode of interaction between Met and the p66 protein isoform of Shc and its effects on rewiring binding effector complexes according to the activation state of the receptor.