Zinc-finger Binding Domain Research Articles

EPEN-22. ZFTA FUSION ONCOPROTEINS DRIVE ONCOGENESIS THROUGH ASSEMBLY OF BIOMOLECULAR CONDENSATES

Abstract BACKGROUND Ependymomas continue to be an aggressive brain tumor entity without targeted therapies. The highly recurrent fusion oncoprotein (FO), ZFTA-RELA, is known to be associated with about 70% of supratentorial ependymomas. While recent studies have highlighted that the fusion protein is involved in aberrant DNA binding and subsequent oncogenic gene expression, the precise mechanisms underlying the transcriptional dysregulation remain elusive. METHODS Using super-resolution and lattice-sheet microscopy, we demonstrate that ZFTA-RELA forms dynamic liquid-like nuclear condensates. ZFTA-RELA condensates recruit co-factors like Med1, and Brd4 and are associated with the transcription of ependymoma oncogenes (Notch1 and Ccnd1). We leveraged machine learning models trained on FOs in other cancers to identify critical intrinsically disordered domains (IDRs) within ZFTA-RELA. A series of mutagenesis experiments were conducted to determine IDRs important for condensate formation, transcription, and tumor initiation. We also performed NMR spectroscopy to unravel the protein structure of ZFTA-RELA zinc finger binding domains. RESULTS We demonstrate that transcriptional condensate formation of ZFTA-RELA is an important cellular phenotype that is tightly linked with oncogenic transcription and tumor initiation. Further, we identify key residues within the zinc finger domain of ZFTA that are required for DNA binding of oncogenic target genes and condensate formation. Deletion of the key IDRs within ZFTA-RELA disrupts condensates and abolishes transcriptional activation and tumorigenesis. We also show that condensate formation is an underlying feature found across all other ZFTA-fusion oncoproteins, providing a model to understand how ZFTA FOs drive cancer. CONCLUSION Our studies provide mechanistic insights into the biophysical and structural basis of ZFTA fusion oncoproteins. These findings provide a framework to develop druggable strategies through targeting ZFTA FO zinc finger domains or disruption of oncogenic condensates.

Genome-Wide Identification and Expression Analysis of GATA Family Genes in Dimocarpus longan Lour.

GATA transcription factors, which are DNA-binding proteins with type IV zinc finger binding domains, have a role in transcriptional regulation in biological organisms. They have an indispensable role in the growth and development of plants, as well as in improvements in their ability to face various environmental stresses. To date, GATAs have been identified in many gene families, but the GATA gene in longan (Dimocarpus longan Lour) has not been studied in previous explorations. Various aspects of genes in the longan GATA family, including their identification and classification, the distribution of their positions on chromosomes, their exon/intron structures, a synteny analysis, their expression at different temperatures, concentration of PEG, early developmental stages of somatic embryos and their expression levels in different tissues, and concentrations of exogenous hormones, were investigated in this study. This study showed that the 22 DlGATAs could be divided into four subfamilies. There were 10 pairs of homologous GATA genes in the synteny analysis of DlGATA and AtGATA. Four segmental replication motifs and one pair of tandem duplication events were present among the DlGATA family members. The cis-acting elements located in promoter regions were also found to be enriched with light-responsive elements, which contained related hormone-responsive elements. In somatic embryos, DlGATA4 is upregulated for expression at the globular embryo (GE) stage. We also found that DlGATA expression was strongly up-regulated in roots and stems. The study demonstrated the expression of DlGATA under hormone (ABA and IAA) treatments in embryogenic callus of longan. Under ABA treatment, DlGATA4 was up-regulated and the other DlGATA genes did not respond significantly. Moreover, as demonstrated with qRT-PCR, the expression of DlGATA genes showed strong up-regulated expression levels under 100 μmol·L-1 concentration IAA treatment. This experiment further studied these and simulated their possible connections with a drought response mechanism, while correlating them with their expression under PEG treatment. Overall, this experiment explored the GATA genes and dug into their evolution, structure, function, and expression profile, thus providing more information for a more in-depth study of the characteristics of the GATA family of genes.

Unveiling Cas8 dynamics and regulation of a transposon-encoded cascade-TniQ complex.

Csy (CRISPR-System Yersinia, or ‘Cascade’) is a class 1, type 1F CRISPR-Cas system which utilizes RNA-guided strand invasion to target viral DNA and designate it for cleavage by an external nuclease (Cas2/3). It has been recently shown, in Vibrio Cholerae, that there exists a variant of Cascade encoded in a transposon (Tn6677). This variant binds to a TniQ homodimer, resulting in a ‘Cascade-TniQ’ complex which, unlike the original Cascade that targets viral DNA for cleavage, recruits a heteromeric transposase responsible for integration of Tn6677. This integration process starts with binding of Cascade's crRNA to the DNA target sequence located in the bacterial genome, resulting in the formation of the ‘R Loop,’ this step is the focus of the current research. Both classical molecular dynamics (cMD) and enhanced sampling methods (GaMD) have been employed to better understand the dynamical state of the system in the RNA-only and the DNA-bound configurations. We have found evidence for a multi-state like behavior, with drastic differences in the state population and transition barriers upon DNA binding. Furthermore, it appears that the TniQ homodimer undergoes changes in large-scale conformations dependent on its level of intrinsic stability. This allowed us to make a tentative claim on a role of Zinc-coordination within TniQ's Zinc-finger binding domains. Tying our findings together with data from literature allowed us to make general comments on how the RNA-bound state and R Loop formation may be regulated. Hopefully, these findings will eventually allow for a more streamlined effort in re-engineering Cascade-TniQ for use in mammalian cells.

Chemical genetics and proteome-wide site mapping reveal cysteine MARylation by PARP-7 on immune-relevant protein targets.

Poly(ADP-ribose) polymerase 7 (PARP-7) has emerged as a critically important member of a large enzyme family that catalyzes ADP-ribosylation in mammalian cells. PARP-7 is a critical regulator of the innate immune response. What remains unclear is the mechanism by which PARP-7 regulates this process, namely because the protein targets of PARP-7 mono-ADP-ribosylation (MARylation) are largely unknown. Here, we combine chemical genetics, proximity labeling, and proteome-wide amino acid ADP-ribosylation site profiling for identifying the direct targets and sites of PARP-7-mediated MARylation in a cellular context. We found that the inactive PARP family member, PARP-13-a critical regulator of the antiviral innate immune response-is a major target of PARP-7. PARP-13 is preferentially MARylated on cysteine residues in its RNA binding zinc finger domain. Proteome-wide ADP-ribosylation analysis reveals cysteine as a major MARylation acceptor of PARP-7. This study provides insight into PARP-7 targeting and MARylation site preference.

Genetic Heterogeneity of KLF1, a Master Regulator of Erythropoiesis, Revealed an Autosomal Recessive Ψβ-Thalassemia and a Very Strong Promoter In Vivo

The Erythroid Kruppel-like Factor 1 or KLF1 is a transcription factor that functioned in the early stage genetic programming of erythroid progenitors to promote physiological γ to β globin gene switching. Indeed, we showed that a truncation mutation (p.K288X) in KLF1 that deleted the DNA binding zinc finger domain resulted in marked KLF1 deficiency and a hematological condition that resembled a hereditary persistence of fetal hemoglobin (HbF) or a β thalassemia. Here, we describe five additional families with the same p.K288X mutation but varied hematological and HbF levels together with genetic and phenotypic data on a 600 data-set from the same Maltese population. The data accounted for a strong promoter embedded within a region of genetic heterogeneity of KLF1 that led to a ψβ thalassemia. Whole genome sequencing on 15 subjects of six families (FamF1 - FamF6) segregating (two) p.K288X frameworks of KLF1 had variable degrees of microcytosis (MCV; 76.1fL -77.4 fL) and HbF levels (HbF 2480 mg/dL - 802mg/dL) due to complex heterozygosity between promoter, coding and truncating mutations in KLF1. Case II-6 of FamF1 with the highest HbF (2480 mg/dL) had 2 promoter and 2 coding mutations in cis and in trans to the p.K288X truncation. Nine (9) KLF1 frameworks (A - I) were derived by transmission disequilibrium analyses of the family data, each assembled from 15 mutations and resulting in 7 genotypes among the families. The p.K288X truncation was found on a second rarer framework. Additional, rarer KLF1 frameworks were found with haploview in the population dataset. The population dataset was made up of 198 β thalassemia heterozygotes and 400 others from the clinic and the biobank and that had no β globin gene mutations, variable blood counts or hemoglobin profiles or both. They were older than 2 years of age, not pregnant and had normal iron levels. The number of KLF1 mutations differed from 0 in the wild-type framework A to 6 in one of the rare frameworks X. Six mutations were in the promoter region and 6 were in the coding region that defined a "KLF1 Variable Region" 5' genomic coordinate 12887183 - 12888273, whereas very few were found in the 3' (genomic coordinate 12884589 - 12884752) that defined the KLF1 "Constant Region" The Constant region has also been evolutionary conserved. It included the zinc finger domain and the proteasome binding site. The genotype - phenotype correlations and the family data were consistent with an autosomal recessive condition that resembled a β thalassemia, thus a ψβ thalassemia. It differed from a silent thalassemia because the β globin gene sequence was wild type. It provided a diagnosis for families with iron resistant microcytosis and borderline hemoglobin phenotypes suitable for counselling in the clinical setting. It was consistent with the observation among the families regarding the high strength of the KLF1 promoter. Multiple "hits" were necessary to suppress the biosynthesis of KLF1 for hemoglobin switching to escape perinatal suppression. The effect of the 6 promoter mutations were confirmed in vitro with native and induced K562 and Hek293T cell lines. Presumably, during normal development, the strong promoter served to rapidly drown KLF1 binding sites with KLF1 molecules to direct progenitors to erythropoiesis with the appropriate adult hemoglobin profile in the perinatal period. The diagnosis of the patients with selected genotypes due to compound and double heterozygosities in promoter and coding sequences shall further permit quantification of differential promoter function in vivo. Figure Disclosures No relevant conflicts of interest to declare.

A retrotransposon gag-like-3 gene RTL3 and SOX-9 co-regulate the expression of COL2A1 in chondrocytes

ABSTRACT Purpose Transposable elements are known to remodel gene structure and provide a known source of genetic variation. Retrotransposon gag-like-3 (RTL3) is a mammalian retrotransposon-derived transcript (MART) whose function in the skeletal tissue is unknown. This study aimed to elucidate the biological significance of RTL3 in chondrogenesis and type-II collagen (COL2A1) gene expression in chondrocytes. Materials and Methods Expression of RTL3, SOX-9 and COL2A1 mRNAs was determined by TaqMan assays and the protein expression by immunoblotting. RTL3 and Sox-9 depletion in human chondrocytes was achieved using validated siRNAs. An RTL3 mutant (∆RTL3) lacking the zinc finger domain was created using in vitro mutagenesis. Forced expression of RTL3, ∆RTL3, and SOX-9 was achieved using CMV promoter containing expression plasmids. CRISPR-Cas9 was utilized to delete Rtl3 and create a stable ATDC5Rlt3-/- cell line. Matrix deposition and Col2a1 quantification during chondrogenesis were determined by Alcian blue staining and Sircol™ Soluble Collagen Assay, respectively. Results RTL3 is not ubiquitously expressed but showed strong expression in cartilage, chondrocytes and synoviocytes but not in muscle, brain, or other tissues analyzed. Loss-of-function and gain-of-function studies demonstrated a critical role of RTL3 in the regulation of SOX-9 and COL2A1 expression and matrix synthesis during chondrogenesis. Both RTL3 and SOX-9 displayed co-regulated expression in chondrocytes. Gene regulatory activity of RTL3 requires the c-terminal CCHC zinc-finger binding domain. Conclusions Our results identify a novel regulatory mechanism of COL2A1 expression in chondrocytes that may help to further understand the skeletal development and the pathogenesis of diseases with altered COL2A1 expression.

Discovery, characterisation and genomic variation of six novel Gammapapillomavirus types from penile swabs in South Africa

Six novel human papillomaviruses from penile swabs were characterised. Multiple full genome clones for each novel type were generated, and complete genome sizes were: HPV211 (7253bp), HPV212 (7208bp), HPV213 (7096bp), HPV214 (7357), HPV215 (7186bp) and HPV216 (7233bp). Phylogenetically the novel papillomaviruses all clustered with Gammapapillomaviruses: HPV211 is most closely related to HPV168 (72% identity in the L1 nucleotide sequence) of the Gamma-8 species, HPV212 is most closely related to HPV144 (82.9%) of the Gamma-17 species, HPV213 is most closely related to HPV153 (71.8%) of the Gamma-13 species, HPV214 is most closely related to HPV103 (75.3%) of the Gamma-6 species, HPV215 and HPV216 are most closely related to HPV129 (76.8% and 79.2% respectively) of the Gamma-9 species. The novel HPV types demonstrated the classical genomic organisation of Gammapapillomavirusess, with seven open reading frames (ORFs) encoding five early (E1, E2, E4, E6 and E7) and two late (L1 and L2) proteins. Typical of Gammapapillomavirusess the novel types all lacked the E5 ORF and HPV214 also lacked the E6 ORF. HPV212 had nine unique variants, HPV213 had five and HPV215 had four variants. Conserved domains observed among the novel types are the Zinc finger Binding Domain and PDZ domains. A retinoblastoma binding domain (pRB) binding domain in E7 protein was additionally identified in HPV214. This study expands the knowledge of the rapidly growing Gammapapillomavirus genus.

Plant organellar DNA primase-helicase synthesizes RNA primers for organellar DNA polymerases using a unique recognition sequence.

DNA primases recognize single-stranded DNA (ssDNA) sequences to synthesize RNA primers during lagging-strand replication. Arabidopsis thaliana encodes an ortholog of the DNA primase-helicase from bacteriophage T7, dubbed AtTwinkle, that localizes in chloroplasts and mitochondria. Herein, we report that AtTwinkle synthesizes RNA primers from a 5′-(G/C)GGA-3′ template sequence. Within this sequence, the underlined nucleotides are cryptic, meaning that they are essential for template recognition but are not instructional during RNA synthesis. Thus, in contrast to all primases characterized to date, the sequence recognized by AtTwinkle requires two nucleotides (5′-GA-3′) as a cryptic element. The divergent zinc finger binding domain (ZBD) of the primase module of AtTwinkle may be responsible for template sequence recognition. During oligoribonucleotide synthesis, AtTwinkle shows a strong preference for rCTP as its initial ribonucleotide and a moderate preference for rGMP or rCMP incorporation during elongation. RNA products synthetized by AtTwinkle are efficiently used as primers for plant organellar DNA polymerases. In sum, our data strongly suggest that AtTwinkle primes organellar DNA polymerases during lagging strand synthesis in plant mitochondria and chloroplast following a primase-mediated mechanism. This mechanism contrasts to lagging-strand DNA replication in metazoan mitochondria, in which transcripts synthesized by mitochondrial RNA polymerase prime mitochondrial DNA polymerase γ.

Variation in the Zinc Finger of PRDM9 is Associated with the Absence of Recombination along Nondisjoined Chromosomes 21 of Maternal Origin.

Variation in the zinc finger-binding domain (ZFBD) of the protein PR Domain-Containing Protein 9 (PRDM9) is associated with altered placement of recombination in the human genome. As both the absence and altered placement of recombination are observed among chromosomes 21 that nondisjoin, we genotyped the PRDM9 ZFBD among mothers of children with Trisomy 21 in efforts to determine if variation within this region is associated with the recombination-related risk for chromosome 21 nondisjunction (NDJ). In our approach, PCR was used to amplify the ZFBD of PRDM9 and products were then subjected to bi-directional Sanger sequencing. DNA sequencing reads were aligned and compared to the sequence of the PRDM9 alleles previously identified. Chi-Square analysis was used to compare allele frequencies between cases (N=235, mothers of children with maternally-derived Trisomy 21) and controls (N=48, fathers of children with maternally-derived Trisomy 21). Results of our analysis showed that the frequency of PRDM9 ZF minor alleles is significantly increased among women displaying NDJ of chromosome 21 and no recombination on 21q (p=0.02). Even more, when compared to those for the PRDM9 major A-allele, these minor alleles displayed fewer predicted binding sites on 21q. These findings suggest that allelic variation in the ZF of PRDM9 may play a role in the risk for chromosome 21 NDJ by leading to reduced recombination on 21q.

Bio-informatics analysis of renal carcinoma gene matrix metalloproteinase-7

Renal cancer is one of the common malignant tumors of the urinary system, seriously threatening human being's health. The current discoveries, however, are far enough for efficient and secure treatment of renal cancer. The aim was to explore the mechanism of matrix metalloproteinase-7 (MMP-7) protein in renal carcinoma cell metastasis by bioinformatics analysis. Bioinformatics methods were used to analyze the composition of amino acids, as well as transmembrane structure, coiled coils, subcellular localization, signal peptide, functions and structures at all levels. It showed that the gene MMP-7 totally had 1131 bp. A peptide chain containing 267 amino acids was encoded in the coding region. Based on random coil, α helix, and further super-helix, it had formed a stable neutral hydrophilic protein. The subcellular location analysis indicated that the protein was located outside the cell. The mature peptide started from the 18th amino acid, and its front-end was the sequence of the signal peptide, belonging to the secreted protein. Analysis of the functional domain showed that this protein had two functional domains, the PG binding domain, and the zinc finger binding domain. Moreover, the protein, which was cross-linked with it, was also one related to cancer cell proliferation and metastasis. To sum up, MMP-7 is a stable neutral hydrophilic secreted protein, and it may play a vital role in the invasion and metastasis of cancer cells.

Androgen receptor (AR) suppresses normal human prostate epithelial cell proliferation via AR/β-catenin/TCF-4 complex inhibition of c-MYC transcription.

Physiologic testosterone continuously stimulates prostate stromal cell secretion of paracrine growth factors (PGFs), which if unopposed would induce hyperplastic overgrowth of normal prostate epithelial cells (PrECs). Lentiviral shRNA stable knock down of c-MYC, β-catenin, or TCF-4 completely inhibits normal (i.e., non-transformed) human PrECs growth. c-MYC enhancer driven reporter expression and growth is inhibited by two chemically distinct molecules, which prevent β-catenin signaling either by blocking TCF-4 binding (i.e., toxoflavin) or by stimulating degradation (i.e., AVX939). Recombinant DKK1 protein at a dose, which inhibits activation of canonical Wnt signaling does not inhibit PrEC growth. Nuclear β-catenin translocation and PrEC growth is prevented by both lack of PGFs or Akt inhibitor-I. Growth inhibition induced by lack of PGFs, toxoflavin, or Akt inhibitor-I is overcome by constitutive c-MYC transcription. In the presence of continuous PGF signaling, PrEC hyperplasia is prevented by androgen binding to AR suppressing c-MYC transcription, resulting in G0 arrest/terminal differentiation independent of Rb, p21, p27, FoxP3, or down regulation of growth factors receptors and instead involves androgen-induced formation of AR/β-catenin/TCF-4 complexes, which suppress c-MYC transcription. Such suppression does not occur when AR is mutated in its zinc-finger binding domain. Proliferation of non-transformed human PrECs is dependent upon c-MYC transcription via formation/binding of β-catenin/TCF-4 complexes at both 5' and 3' c-MYC enhancers stimulated by Wnt-independent, PGF induced Akt signaling. In the presence of continuous PGF signaling, PrEC hyperplasia is prevented by androgen-induced formation of AR/β-catenin/TCF-4 complexes, which retains binding to 3' c-MYC enhancer, but now suppresses c-MYC transcription.

Probing the Unfolding of Myoglobin and Domain C of PARP-1 with Covalent Labeling and Top-Down Ultraviolet Photodissociation Mass Spectrometry

Ultraviolet photodissocation (UVPD) mass spectrometry was used for high mass accuracy top-down characterization of two proteins labeled by the chemical probe, S-ethylacetimidate (SETA), in order to evaluate conformational changes as a function of denaturation. The SETA labeling/UVPD-MS methodology was used to monitor the mild denaturation of horse heart myoglobin by acetonitrile, and the results showed good agreement with known acetonitrile and acid unfolding pathways of myoglobin. UVPD outperformed electron transfer dissociation (ETD) in terms of sequence coverage, allowing the SETA reactivity of greater number of lysine amines to be monitored and thus providing a more detailed map of myoglobin. This strategy was applied to the third zinc-finger binding domain, domain C, of PARP-1 (PARP-C), to evaluate the discrepancies between the NMR and crystal structures which reported monomer and dimer forms of the protein, respectively. The trends reflected from the reactivity of each lysine as a function of acetonitrile denaturation in the present study support that PARP-C exists as a monomer in solution with a close-packed C-terminal α helix. Additionally, those lysines for which the SETA reactivity increased under denaturing conditions were found to engage in tertiary polar contacts such as salt bridging and hydrogen bonding, providing evidence that the SETA/UVPD-MS approach offers a versatile means to probe the interactions responsible for conformational changes in proteins.

The E3 ubiquitin ligases RNF126 and Rabring7 regulate endosomal sorting of the Epidermal Growth Factor Receptor

Activation of the epidermal growth factor receptor (EGFR) results in internalization and ubiquitin-dependent endosomal sorting, leading to lysosomal degradation. Here we describe the role of the RING-finger-domain-containing protein RNF126 and the related protein, Rabring7 in EGFR endosomal sorting. We demonstrate that RNF126 specifies K48-linked chains with UbcH5b and also functions with Ubc13/Uev1a to form K63-linked chains in vitro. RNF126 and Rabring7 associate with the EGFR through a ubiquitin-binding zinc finger domain and both E3 ubiquitin ligases promote ubiquitylation of EGFR. In the absence of c-Cbl or in cells expressing Cbl-70Z, the binding of RNF126 and Rabring7 to the EGFR is reduced, suggesting that RNF126 and Rabring7 function downstream of c-Cbl. In HeLa cells depleted of either RNF126 or Rabring7 the EGFR is retained in a late endocytic compartment and is inefficiently degraded. In addition, depletion of RNF126 or Rabring7 destabilizes ESCRT-II and reduces the number of multivesicular bodies formed after EGF stimulation. We also show that the depletion of Rabring7 attenuates the degradation of MET and that both RNF126 and Rabring7 regulate the sorting of CXCR4 from an early endocytic compartment. Together these data suggest that RNF126 and Rabring7 play a role in the ubiquitin-dependent sorting and downregulation of membrane receptors.

Climbing STAIRs towards clinical trials with a novel PARP-1 inhibitor for the treatment of ischemic stroke

Climbing STAIRs towards clinical trials with a novel PARP-1 inhibitor for the treatment of ischemic stroke

Interruption of intrachromosomal looping by CCCTC binding factor decoy proteins abrogates genomic imprinting of human insulin-like growth factor II

Monoallelic expression of IGF2 is regulated by CCCTC binding factor (CTCF) binding to the imprinting control region (ICR) on the maternal allele, with subsequent formation of an intrachromosomal loop to the promoter region. The N-terminal domain of CTCF interacts with SUZ12, part of the polycomb repressive complex-2 (PRC2), to silence the maternal allele. We synthesized decoy CTCF proteins, fusing the CTCF deoxyribonucleic acid-binding zinc finger domain to CpG methyltransferase Sss1 or to enhanced green fluorescent protein. In normal human fibroblasts and breast cancer MCF7 cell lines, the CTCF decoy proteins bound to the unmethylated ICR and to the IGF2 promoter region but did not interact with SUZ12. EZH2, another part of PRC2, was unable to methylate histone H3-K27 in the IGF2 promoter region, resulting in reactivation of the imprinted allele. The intrachromosomal loop between the maternal ICR and the IGF2 promoters was not observed when IGF2 imprinting was lost. CTCF epigenetically governs allelic gene expression of IGF2 by orchestrating chromatin loop structures involving PRC2.

Selective modulation of the redox state and thus the repressor function of SLUG by peroxiredoxin 5 at the BRCA2 gene silencer in human breast cancer cells

Human BRCA2 gene expression is inhibited by the transcriptional repressor protein SLUG at the G0/G1 phase of SLUG‐high breast cancer cells through the binding of SLUG to an E2‐box sequence in the BRCA2 gene. Interestingly, while SLUG maintains its repression of several of its target genes at the S/G2 phase of cell growth, it fails to repress BRCA2 gene at this growth phase. We discovered that, unlike other SLUG target gene promoters, BRCA2 gene silencer has the SLUG‐binding E2‐box at the close proximity of the binding site of the redox modulator protein peroxiredoxin 5 (PRDX5). Here, we provide evidence that in the S/G2 phase of cell growth, oxidized PRDX5 enters the nucleus at a higher rate and binds to BRCA2 silencer to rip off SLUG from its E2‐box through oxidation of its DNA binding zinc finger domains resulting in the de‐silencing of BRCA2 gene expression. This study presents a novel modality of PRDX5 in site‐specific oxidation of zinc‐finger transcription factors to regulate gene expression in a redox‐dependent manner. Supported by DOD‐CDMRP BCRP Grants W81XWH‐06‐1‐0466, W81XWH‐08‐1‐0446, BC086542, and Susan G. Komen Breast Cancer Foundation grant# BCTR0707627 to GC and 1U54RR026140‐01to SM.

Gene Correction in Human Embryonic and Induced Pluripotent Stem Cells: Promises and Challenges Ahead

The sequencing of the human genome has greatly facilitated our ability to identify the gene candidates that are critical for developmental regulation and disease progression. However, investigators seeking to use such data to gain a better understanding of the developmental biology and therapeutic potential of pluripotent stem cell types may be frustrated by the limitations of current genetic engineering techniques. Targeted genomic manipulation of mammalian cells is inefficient, and unintended effects of such manipulation on the cells’ developmental and proliferative potential remain uninvestigated. To this end, the recent demonstration of targeted gene correction in human induced pluripotent stem cells (hiPSCs) before and after reprogramming, as reported in this issue of Molecular Therapy, warrants special mention.1

DNA Replication-Coupled PCNA Mono-Ubiquitination and Polymerase Switching in a Human InVitro System

Translesion DNA synthesis is a mechanism of DNA damage tolerance, and mono-ubiquitination of proliferating cell nuclear antigen (PCNA) is considered to play a key role in regulating the switch from replicative to translesion DNA polymerases (pols). In this study, we analyzed effects of a replicative pol δ on PCNA mono-ubiquitination with the ubiquitin-conjugating enzyme and ligase UBE2A/HHR6A/RAD6A–RAD18. The results revealed that PCNA interacting with pol δ is a better target for ubiquitination, and PCNA mono-ubiquitination could be coupled with DNA replication. Consequently, we could reconstitute replication-coupled switching between pol δ and a translesion pol, pol η, on an ultraviolet-light-irradiated template. With this system, we obtained direct evidence that polymerase switching reactions are stimulated by mono-ubiquitination of PCNA, depending on a function of the ubiquitin binding zinc finger domain of pol η. This study provides a framework for detailed analyses of molecular mechanisms of human pol switching and regulation of translesion DNA synthesis.

The structural role of the zinc ion can be dispensable in prokaryotic zinc-finger domains

The recent characterization of the prokaryotic Cys(2)His(2) zinc-finger domain, identified in Ros protein from Agrobacterium tumefaciens, has demonstrated that, although possessing a similar zinc coordination sphere, this domain is structurally very different from its eukaryotic counterpart. A search in the databases has identified approximately 300 homologues with a high sequence identity to the Ros protein, including the amino acids that form the extensive hydrophobic core in Ros. Surprisingly, the Cys(2)His(2) zinc coordination sphere is generally poorly conserved in the Ros homologues, raising the question of whether the zinc ion is always preserved in these proteins. Here, we present a functional and structural study of a point mutant of Ros protein, Ros(56-142)C82D, in which the second coordinating cysteine is replaced by an aspartate, 5 previously-uncharacterized representative Ros homologues from Mesorhizobium loti, and 2 mutants of the homologues. Our results indicate that the prokaryotic zinc-finger domain, which in Ros protein tetrahedrally coordinates Zn(II) through the typical Cys(2)His(2) coordination, in Ros homologues can either exploit a CysAspHis(2) coordination sphere, previously never described in DNA binding zinc finger domains to our knowledge, or lose the metal, while still preserving the DNA-binding activity. We demonstrate that this class of prokaryotic zinc-finger domains is structurally very adaptable, and surprisingly single mutations can transform a zinc-binding domain into a nonzinc-binding domain and vice versa, without affecting the DNA-binding ability. In light of our findings an evolutionary link between the prokaryotic and eukaryotic zinc-finger domains, based on bacteria-to-eukaryota horizontal gene transfer, is discussed.

GLI2-specific Transcriptional Activation of the Bone Morphogenetic Protein/Activin Antagonist Follistatin in Human Epidermal Cells

Hedgehog (HH) signaling in the epidermis is primarily mediated by the zinc finger transcription factors GLI1 and GLI2. Exquisite regulation of HH/GLI signaling is crucial for proper specification of the epidermal lineage and development of its derivatives, whereas dysregulation of HH/GLI signaling disrupts tissue homeostasis and causes basal cell carcinoma (BCC). Similarly, bone morphogenetic proteins (BMPs) and activins have been described as key signaling factors in the complex regulation of epidermal fate decisions, although their precise interplay with HH/GLI is largely elusive. Here we show that, in human epidermal cells, expression of the activin/BMP antagonist follistatin (FST) is predominantly up-regulated by the HH effector GLI2. Consistently, we found strong FST expression in the outer root sheath of human hair follicles and BCC. Detailed promoter analysis showed that two sequences with homology to the GLI consensus binding site are required for GLI2-mediated activation. Interestingly, activation of the FST promoter is highly GLI2-specific, because neither GLI1 nor GLI3 can significantly increase FST transcription. GLI2 specificity requires the presence of a 518-bp fragment in the proximal FST promoter region. On the protein level, sequences C-terminal to the zinc finger are responsible for GLI2-specific activation of FST transcription, pointing to the existence of GLI-interacting cofactors that modulate GLI target specificity. Our results reveal a key role of GLI2 in activation of the activin/BMP antagonist FST in response to HH signaling and provide new evidence for a regulatory interaction between HH and activin/BMP signaling in hair follicle development and BCC.